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8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 153
Recombinant-DNATechnolo
Manipulating DNA molecules
8172019 Restriction Enzymes and Vectors
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Objectives
ndashthinsp
Describe Restriction Endonucleases (RE)
ndashthinsp Understand their biological role
ndashthinsp Understand their nomenclature and classification
ndashthinsp Describe their general mode of actionoperation
ndashthinsp Understand the importance of RErsquos tondashthinsp Recombinant DNA (R-DNA) Technology
8172019 Restriction Enzymes and Vectors
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Cloning
Strategy
Preparation of VectorPreparation of Vector
Preparation of InsertPreparation of Insert
Ligation ReactionLigation Reaction
SelectionSelection
Isolation of RIsolation of R--DNADNA(plasmid(plasmid prepsetcprepsetc))
Sequence and TransformSequence and Transform
8172019 Restriction Enzymes and Vectors
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P
P P
P
DNA fragmentVector
tetRAmpR
DNA ligase
- Recombinant DNA- Recombinant vector- DNA chimera
Cut with restrictionendonuclease
8172019 Restriction Enzymes and Vectors
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Recombinant-DNA technology1 Restriction digestion
2 Joining DNA molecules3 Transformation
4 Selection Screening = identifying-
DNA molecule5 Cloning
8172019 Restriction Enzymes and Vectors
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Restriction endonucleases
bull Also called restriction enzymes
bull Restriction endonucleases enzymes found in bacteria capable ofspecifically binding to DNA and cleaving dsDNA
bull Restriction endonucleases along with DNA Methylases(modifying enzymes) form a bacterial defense system againstbacteriophages
8172019 Restriction Enzymes and Vectors
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Restriction Endonucleases- defense function
bull Methyltransferases or methylases add a methyl group to DNA in
some bacteria
bull Restriction endonucleases will not cleave at their restriction siteif it is methylated
bull Invading DNA usually not methylated will be cleaved and thusinactivated by the bacteria whose own genome is methylated
8172019 Restriction Enzymes and Vectors
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8172019 Restriction Enzymes and Vectors
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Restriction Endonucleases - three typestype I II and III
8172019 Restriction Enzymes and Vectors
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Restriction Endonucleases
R-DNA technology uses type II ndash Why
1 Restriction activity and modifying activity are in separateenzymes
2 Restriction endonucleases cleave within the recognition site oradjacent to it
3 Do not need ATP for activity Needs only Mg++
4 Cuts in a precise manner very predictable
5 Short palindromic recognition sequence
Note
Over 1500 known over 150 available commercially
8172019 Restriction Enzymes and Vectors
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Restriction Endonucleases- Nomenclature
Named for bacterial genus species strain and type
xamp e coGenus E scherichia Species co li Strain ROrder discovered 1
8172019 Restriction Enzymes and Vectors
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Restriction Endonucleases- Nomenclature
eg Bam H1Bacillus amyloliquefaciens Strain H
First enzyme characterised
eg Hin dIIIHaemophilus influenzae Strain D - 3rd enzyme
eg Eco RI
Escherichia coli Strain R ndash 1st enzyme discovered
8172019 Restriction Enzymes and Vectors
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Restriction Endonucleases
Recognition sites have symmetry (palindromic)
ldquoAble was I ere I saw Elbardquo
Bam H1 site5rsquo-GGATCC-3rsquo
3rsquo-CCTAGG-5rsquo
8172019 Restriction Enzymes and Vectors
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Restriction Endonucleases
eg Bam H1
Cleavage site
GGATCCCCTAGG
Recognition sequence
8172019 Restriction Enzymes and Vectors
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Restriction Endonucleases
Cohesive end cutter or sticky end cutter
8172019 Restriction Enzymes and Vectors
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Restriction Endonucleases
eg Sma I = Serratia marcescens Enzyme 1
CCCGGG
Cleavage site
GGGCCC
Recognition sequence
BLUNT END CUTTEReg Sca1 EcoRV Nru1
8172019 Restriction Enzymes and Vectors
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Classification of Restriction Endonucleases
1 Sticky-end cutter vs blunt-end cutter
2 Frequent cutter vs rare cutter
Recognition sequence Cleavage frequency
4 bp (Alu1 HpaII HhaI) (144) = 1256
5 bp (Hga1) (145 ) = 110246 bp (BamH1 HinD111) (146 ) = 14096
7 bp (PpuM1) (147 ) = 116384
8 bp (Not1)
9 bp (AlwN1)10 bp
8172019 Restriction Enzymes and Vectors
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983122983141983139983151983149983138983145983150983137983150983156 983140983150983137 983154983141983155983156983154983145983139983156983145983151983150
983141983150983140983151983150983157983139983148983141983137983155983141983155
8172019 Restriction Enzymes and Vectors
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Classification of Restriction Endonucleases
3 Ambiguous vs precise cutters
eg BsiE1
CGPuPyCGGCPyPuGC
8172019 Restriction Enzymes and Vectors
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Classification of Restriction Endonucleases
4 Isoshizomers
Some enzymes have the same target site (recognition site)
Two subtypes
Same target site ndash different cleavage site
eg Sma1 and Xma1
Same target site - same cleavage site
eg Aat1 and Stu1
BamH1 and Bst1
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8172019 Restriction Enzymes and Vectors
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8172019 Restriction Enzymes and Vectors
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Restriction digestionCommercial restriction enzymes
1 Restriction enzymes are commercially available at 5 units per
microL concentration
2 I unit = 1 unit (U) is the amount if enzyme that catalyses the
conditions
3 Enzymes come in storage buffer containing EDTA DTT BSAand Glycerol
4 Too much enzyme in the reaction mix can cause star activity
8172019 Restriction Enzymes and Vectors
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Star activity
Loss of specificity of the restriction enzyme (typeII) underextreme non-standard conditions is called star activity
Under such conditions the restriction enzyme may cleave similarbut non identical target sequences
Non-standard conditions include1 high glycerol concentration gt5 vv
2 gt100 units per ug of DNA
3 Low ionic strength buffer lt25mM (usually is 100-150 mM)
4 high pH (gt 80) usually pH is around 75 presence of organic solvents DMSO ethanol PEGdimethyl acetamide
6 Cations other than Mg++ eg Cu++ Co++Zn++ Mn++
8172019 Restriction Enzymes and Vectors
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Restriction digestion
Stopping the Restriction digestion
1 Heat inactivation of restriction enzymes
Place reaction mix in a waterbath set at 65oCNote not all Restriction enzymes can be heat inactivated egPvu1 Bgl II BamH1 Acc1
2 Add 2ul of 25 mM EDTAEDTA chelates the Mg++ and makes it unavailable to theRestriction Endonuclease
3 Phenol-chloroform extraction purificationRemoves all proteins including the enzymes
8172019 Restriction Enzymes and Vectors
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EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Single Digests
EcoRI BamHI NcoI
Number offragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
8172019 Restriction Enzymes and Vectors
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Digestions
4kbp
L a d d e r
E c o R I
B a m H I
U n d i g e s t e d
N c o I
EcoRI BamHI NcoI
Numberof
fragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2853
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Double Digests
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
8172019 Restriction Enzymes and Vectors
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Digestions
4kbp
L a d d e r
E c o R I
B a m H I
E c o R I
N c o I
U n d i g e s t e d
B a m H I
N c o I
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3053
SummaryRestriction Digestion is the process of cutting DNA molecules into smaller pieces
with special enzymes called Restriction Endonucleases (sometimes just called
Restriction Enzymes or REs) These special enzymes recognize specificsequences in the DNA molecule (for example GATATC) wherever that sequenceoccurs in the DNA
httphigheredmcgraw-hillcomsites9834092339student_view0chapter18restriction_endonucleaseshtml
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Vectors
8172019 Restriction Enzymes and Vectors
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So what are vectors
bull Vector (molecular biology) vehicle used to transfer genetic material to a target
cell
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So what are vectors
ndash Plasmid vector
ndash Binary vector a cloning vector used to generate transgenic plants
ndash Cloning vector
ndash Expression vector a plasmid specifically used for protein expression in thetarget
ndash Shuttle vector a vector (usually a plasmid) constructed so that it canpropagate in two different host species
ndash Viral vector a virus modified to deliver foreign genetic material into a cell
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Vectors can be classified based on
bull The purpose of the vector ndash eg cloning library construction ssDNA production mRNA synthesis expression
multipurpose etc
bull Origin of vector ndash Plasmid ndash Bacteriophage ndash Cosmid ndash Phagemid ndash YAC BAC
bull The host organism that the vector is propagated in ndash Bacteria (bacterial origin of replication) ndash Yeast (yeast origin of replication) ndash Shuttle vector (both origins)
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Types of vectors
Vector Target fragment length
Plasmid 0-10 kb (total size up to 15 kb)
Cosmid 10-40 kb
P1 artificial chromosome (PAC) 130-150 kbBacterial artificial chromosome (BAC) About 300 kb
Yeast artificial chromosome (YAC) 200 kb to 2 Mb
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What vectors will I be covering
Vectors
bull Plasmid vectorso Cloning vectors
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Plasmid Vectors
bull Plasmids most commonly used in recombinant DNA technology replicate in E coli
bull They have been engineered to optimize their use as vectors in DNA cloning
ndash To simplify working with plasmids their length is reduced
ndash Most plasmid vectors contain little more than the essential nucleotide
sequences required for their use in DNA cloning
bull Plasmid vectors are usually used as either cloning vectors expression vectorsssDNA vectors or transformation vectors
bull They are not used as library vectors
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Basic Characteristics of Cloning Vectors
Basic characteristics
1 Origin of replication (ori )
ndash An origin of replication is a sequence of DNA at which replication is initiated on achromosome plasmid or virus
bull small DNAs a single origin is sufficient ndash Larger DNAs have many origins and DNA replication is initiated at all of them
bull Determines the vector copy number
bull Determines the plasmids compatibility its ability to replicate in conjunction withanother plasmid within the same bacterial cell
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Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
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Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
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Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
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Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
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Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
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Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
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Expression vectors
Bacterial Artificial Chromosome (BAC)
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Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
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Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
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A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
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Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
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Objectives
ndashthinsp
Describe Restriction Endonucleases (RE)
ndashthinsp Understand their biological role
ndashthinsp Understand their nomenclature and classification
ndashthinsp Describe their general mode of actionoperation
ndashthinsp Understand the importance of RErsquos tondashthinsp Recombinant DNA (R-DNA) Technology
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Cloning
Strategy
Preparation of VectorPreparation of Vector
Preparation of InsertPreparation of Insert
Ligation ReactionLigation Reaction
SelectionSelection
Isolation of RIsolation of R--DNADNA(plasmid(plasmid prepsetcprepsetc))
Sequence and TransformSequence and Transform
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P
P P
P
DNA fragmentVector
tetRAmpR
DNA ligase
- Recombinant DNA- Recombinant vector- DNA chimera
Cut with restrictionendonuclease
8172019 Restriction Enzymes and Vectors
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Recombinant-DNA technology1 Restriction digestion
2 Joining DNA molecules3 Transformation
4 Selection Screening = identifying-
DNA molecule5 Cloning
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Restriction endonucleases
bull Also called restriction enzymes
bull Restriction endonucleases enzymes found in bacteria capable ofspecifically binding to DNA and cleaving dsDNA
bull Restriction endonucleases along with DNA Methylases(modifying enzymes) form a bacterial defense system againstbacteriophages
8172019 Restriction Enzymes and Vectors
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Restriction Endonucleases- defense function
bull Methyltransferases or methylases add a methyl group to DNA in
some bacteria
bull Restriction endonucleases will not cleave at their restriction siteif it is methylated
bull Invading DNA usually not methylated will be cleaved and thusinactivated by the bacteria whose own genome is methylated
8172019 Restriction Enzymes and Vectors
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8172019 Restriction Enzymes and Vectors
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Restriction Endonucleases - three typestype I II and III
8172019 Restriction Enzymes and Vectors
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Restriction Endonucleases
R-DNA technology uses type II ndash Why
1 Restriction activity and modifying activity are in separateenzymes
2 Restriction endonucleases cleave within the recognition site oradjacent to it
3 Do not need ATP for activity Needs only Mg++
4 Cuts in a precise manner very predictable
5 Short palindromic recognition sequence
Note
Over 1500 known over 150 available commercially
8172019 Restriction Enzymes and Vectors
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Restriction Endonucleases- Nomenclature
Named for bacterial genus species strain and type
xamp e coGenus E scherichia Species co li Strain ROrder discovered 1
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Restriction Endonucleases- Nomenclature
eg Bam H1Bacillus amyloliquefaciens Strain H
First enzyme characterised
eg Hin dIIIHaemophilus influenzae Strain D - 3rd enzyme
eg Eco RI
Escherichia coli Strain R ndash 1st enzyme discovered
8172019 Restriction Enzymes and Vectors
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Restriction Endonucleases
Recognition sites have symmetry (palindromic)
ldquoAble was I ere I saw Elbardquo
Bam H1 site5rsquo-GGATCC-3rsquo
3rsquo-CCTAGG-5rsquo
8172019 Restriction Enzymes and Vectors
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Restriction Endonucleases
eg Bam H1
Cleavage site
GGATCCCCTAGG
Recognition sequence
8172019 Restriction Enzymes and Vectors
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Restriction Endonucleases
Cohesive end cutter or sticky end cutter
8172019 Restriction Enzymes and Vectors
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Restriction Endonucleases
eg Sma I = Serratia marcescens Enzyme 1
CCCGGG
Cleavage site
GGGCCC
Recognition sequence
BLUNT END CUTTEReg Sca1 EcoRV Nru1
8172019 Restriction Enzymes and Vectors
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Classification of Restriction Endonucleases
1 Sticky-end cutter vs blunt-end cutter
2 Frequent cutter vs rare cutter
Recognition sequence Cleavage frequency
4 bp (Alu1 HpaII HhaI) (144) = 1256
5 bp (Hga1) (145 ) = 110246 bp (BamH1 HinD111) (146 ) = 14096
7 bp (PpuM1) (147 ) = 116384
8 bp (Not1)
9 bp (AlwN1)10 bp
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983122983141983139983151983149983138983145983150983137983150983156 983140983150983137 983154983141983155983156983154983145983139983156983145983151983150
983141983150983140983151983150983157983139983148983141983137983155983141983155
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Classification of Restriction Endonucleases
3 Ambiguous vs precise cutters
eg BsiE1
CGPuPyCGGCPyPuGC
8172019 Restriction Enzymes and Vectors
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Classification of Restriction Endonucleases
4 Isoshizomers
Some enzymes have the same target site (recognition site)
Two subtypes
Same target site ndash different cleavage site
eg Sma1 and Xma1
Same target site - same cleavage site
eg Aat1 and Stu1
BamH1 and Bst1
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Restriction digestionCommercial restriction enzymes
1 Restriction enzymes are commercially available at 5 units per
microL concentration
2 I unit = 1 unit (U) is the amount if enzyme that catalyses the
conditions
3 Enzymes come in storage buffer containing EDTA DTT BSAand Glycerol
4 Too much enzyme in the reaction mix can cause star activity
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Star activity
Loss of specificity of the restriction enzyme (typeII) underextreme non-standard conditions is called star activity
Under such conditions the restriction enzyme may cleave similarbut non identical target sequences
Non-standard conditions include1 high glycerol concentration gt5 vv
2 gt100 units per ug of DNA
3 Low ionic strength buffer lt25mM (usually is 100-150 mM)
4 high pH (gt 80) usually pH is around 75 presence of organic solvents DMSO ethanol PEGdimethyl acetamide
6 Cations other than Mg++ eg Cu++ Co++Zn++ Mn++
8172019 Restriction Enzymes and Vectors
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Restriction digestion
Stopping the Restriction digestion
1 Heat inactivation of restriction enzymes
Place reaction mix in a waterbath set at 65oCNote not all Restriction enzymes can be heat inactivated egPvu1 Bgl II BamH1 Acc1
2 Add 2ul of 25 mM EDTAEDTA chelates the Mg++ and makes it unavailable to theRestriction Endonuclease
3 Phenol-chloroform extraction purificationRemoves all proteins including the enzymes
8172019 Restriction Enzymes and Vectors
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EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Single Digests
EcoRI BamHI NcoI
Number offragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
8172019 Restriction Enzymes and Vectors
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Digestions
4kbp
L a d d e r
E c o R I
B a m H I
U n d i g e s t e d
N c o I
EcoRI BamHI NcoI
Numberof
fragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
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EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Double Digests
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
8172019 Restriction Enzymes and Vectors
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Digestions
4kbp
L a d d e r
E c o R I
B a m H I
E c o R I
N c o I
U n d i g e s t e d
B a m H I
N c o I
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
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SummaryRestriction Digestion is the process of cutting DNA molecules into smaller pieces
with special enzymes called Restriction Endonucleases (sometimes just called
Restriction Enzymes or REs) These special enzymes recognize specificsequences in the DNA molecule (for example GATATC) wherever that sequenceoccurs in the DNA
httphigheredmcgraw-hillcomsites9834092339student_view0chapter18restriction_endonucleaseshtml
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Vectors
8172019 Restriction Enzymes and Vectors
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So what are vectors
bull Vector (molecular biology) vehicle used to transfer genetic material to a target
cell
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So what are vectors
ndash Plasmid vector
ndash Binary vector a cloning vector used to generate transgenic plants
ndash Cloning vector
ndash Expression vector a plasmid specifically used for protein expression in thetarget
ndash Shuttle vector a vector (usually a plasmid) constructed so that it canpropagate in two different host species
ndash Viral vector a virus modified to deliver foreign genetic material into a cell
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Vectors can be classified based on
bull The purpose of the vector ndash eg cloning library construction ssDNA production mRNA synthesis expression
multipurpose etc
bull Origin of vector ndash Plasmid ndash Bacteriophage ndash Cosmid ndash Phagemid ndash YAC BAC
bull The host organism that the vector is propagated in ndash Bacteria (bacterial origin of replication) ndash Yeast (yeast origin of replication) ndash Shuttle vector (both origins)
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Types of vectors
Vector Target fragment length
Plasmid 0-10 kb (total size up to 15 kb)
Cosmid 10-40 kb
P1 artificial chromosome (PAC) 130-150 kbBacterial artificial chromosome (BAC) About 300 kb
Yeast artificial chromosome (YAC) 200 kb to 2 Mb
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What vectors will I be covering
Vectors
bull Plasmid vectorso Cloning vectors
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Plasmid Vectors
bull Plasmids most commonly used in recombinant DNA technology replicate in E coli
bull They have been engineered to optimize their use as vectors in DNA cloning
ndash To simplify working with plasmids their length is reduced
ndash Most plasmid vectors contain little more than the essential nucleotide
sequences required for their use in DNA cloning
bull Plasmid vectors are usually used as either cloning vectors expression vectorsssDNA vectors or transformation vectors
bull They are not used as library vectors
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Basic Characteristics of Cloning Vectors
Basic characteristics
1 Origin of replication (ori )
ndash An origin of replication is a sequence of DNA at which replication is initiated on achromosome plasmid or virus
bull small DNAs a single origin is sufficient ndash Larger DNAs have many origins and DNA replication is initiated at all of them
bull Determines the vector copy number
bull Determines the plasmids compatibility its ability to replicate in conjunction withanother plasmid within the same bacterial cell
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Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
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Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
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Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
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Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
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Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
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Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
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Expression vectors
Bacterial Artificial Chromosome (BAC)
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Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
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Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
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A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
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Next DayTransformation SelectionScreening
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Cloning
Strategy
Preparation of VectorPreparation of Vector
Preparation of InsertPreparation of Insert
Ligation ReactionLigation Reaction
SelectionSelection
Isolation of RIsolation of R--DNADNA(plasmid(plasmid prepsetcprepsetc))
Sequence and TransformSequence and Transform
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P
P P
P
DNA fragmentVector
tetRAmpR
DNA ligase
- Recombinant DNA- Recombinant vector- DNA chimera
Cut with restrictionendonuclease
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Recombinant-DNA technology1 Restriction digestion
2 Joining DNA molecules3 Transformation
4 Selection Screening = identifying-
DNA molecule5 Cloning
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Restriction endonucleases
bull Also called restriction enzymes
bull Restriction endonucleases enzymes found in bacteria capable ofspecifically binding to DNA and cleaving dsDNA
bull Restriction endonucleases along with DNA Methylases(modifying enzymes) form a bacterial defense system againstbacteriophages
8172019 Restriction Enzymes and Vectors
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Restriction Endonucleases- defense function
bull Methyltransferases or methylases add a methyl group to DNA in
some bacteria
bull Restriction endonucleases will not cleave at their restriction siteif it is methylated
bull Invading DNA usually not methylated will be cleaved and thusinactivated by the bacteria whose own genome is methylated
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Restriction Endonucleases - three typestype I II and III
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Restriction Endonucleases
R-DNA technology uses type II ndash Why
1 Restriction activity and modifying activity are in separateenzymes
2 Restriction endonucleases cleave within the recognition site oradjacent to it
3 Do not need ATP for activity Needs only Mg++
4 Cuts in a precise manner very predictable
5 Short palindromic recognition sequence
Note
Over 1500 known over 150 available commercially
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Restriction Endonucleases- Nomenclature
Named for bacterial genus species strain and type
xamp e coGenus E scherichia Species co li Strain ROrder discovered 1
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Restriction Endonucleases- Nomenclature
eg Bam H1Bacillus amyloliquefaciens Strain H
First enzyme characterised
eg Hin dIIIHaemophilus influenzae Strain D - 3rd enzyme
eg Eco RI
Escherichia coli Strain R ndash 1st enzyme discovered
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Restriction Endonucleases
Recognition sites have symmetry (palindromic)
ldquoAble was I ere I saw Elbardquo
Bam H1 site5rsquo-GGATCC-3rsquo
3rsquo-CCTAGG-5rsquo
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Restriction Endonucleases
eg Bam H1
Cleavage site
GGATCCCCTAGG
Recognition sequence
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Restriction Endonucleases
Cohesive end cutter or sticky end cutter
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Restriction Endonucleases
eg Sma I = Serratia marcescens Enzyme 1
CCCGGG
Cleavage site
GGGCCC
Recognition sequence
BLUNT END CUTTEReg Sca1 EcoRV Nru1
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Classification of Restriction Endonucleases
1 Sticky-end cutter vs blunt-end cutter
2 Frequent cutter vs rare cutter
Recognition sequence Cleavage frequency
4 bp (Alu1 HpaII HhaI) (144) = 1256
5 bp (Hga1) (145 ) = 110246 bp (BamH1 HinD111) (146 ) = 14096
7 bp (PpuM1) (147 ) = 116384
8 bp (Not1)
9 bp (AlwN1)10 bp
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983122983141983139983151983149983138983145983150983137983150983156 983140983150983137 983154983141983155983156983154983145983139983156983145983151983150
983141983150983140983151983150983157983139983148983141983137983155983141983155
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Classification of Restriction Endonucleases
3 Ambiguous vs precise cutters
eg BsiE1
CGPuPyCGGCPyPuGC
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Classification of Restriction Endonucleases
4 Isoshizomers
Some enzymes have the same target site (recognition site)
Two subtypes
Same target site ndash different cleavage site
eg Sma1 and Xma1
Same target site - same cleavage site
eg Aat1 and Stu1
BamH1 and Bst1
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Restriction digestionCommercial restriction enzymes
1 Restriction enzymes are commercially available at 5 units per
microL concentration
2 I unit = 1 unit (U) is the amount if enzyme that catalyses the
conditions
3 Enzymes come in storage buffer containing EDTA DTT BSAand Glycerol
4 Too much enzyme in the reaction mix can cause star activity
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Star activity
Loss of specificity of the restriction enzyme (typeII) underextreme non-standard conditions is called star activity
Under such conditions the restriction enzyme may cleave similarbut non identical target sequences
Non-standard conditions include1 high glycerol concentration gt5 vv
2 gt100 units per ug of DNA
3 Low ionic strength buffer lt25mM (usually is 100-150 mM)
4 high pH (gt 80) usually pH is around 75 presence of organic solvents DMSO ethanol PEGdimethyl acetamide
6 Cations other than Mg++ eg Cu++ Co++Zn++ Mn++
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Restriction digestion
Stopping the Restriction digestion
1 Heat inactivation of restriction enzymes
Place reaction mix in a waterbath set at 65oCNote not all Restriction enzymes can be heat inactivated egPvu1 Bgl II BamH1 Acc1
2 Add 2ul of 25 mM EDTAEDTA chelates the Mg++ and makes it unavailable to theRestriction Endonuclease
3 Phenol-chloroform extraction purificationRemoves all proteins including the enzymes
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EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Single Digests
EcoRI BamHI NcoI
Number offragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
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Digestions
4kbp
L a d d e r
E c o R I
B a m H I
U n d i g e s t e d
N c o I
EcoRI BamHI NcoI
Numberof
fragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
3kbp
2kbp
1kbp
05kbp
025kbp
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EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Double Digests
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
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Digestions
4kbp
L a d d e r
E c o R I
B a m H I
E c o R I
N c o I
U n d i g e s t e d
B a m H I
N c o I
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
3kbp
2kbp
1kbp
05kbp
025kbp
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SummaryRestriction Digestion is the process of cutting DNA molecules into smaller pieces
with special enzymes called Restriction Endonucleases (sometimes just called
Restriction Enzymes or REs) These special enzymes recognize specificsequences in the DNA molecule (for example GATATC) wherever that sequenceoccurs in the DNA
httphigheredmcgraw-hillcomsites9834092339student_view0chapter18restriction_endonucleaseshtml
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Vectors
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So what are vectors
bull Vector (molecular biology) vehicle used to transfer genetic material to a target
cell
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So what are vectors
ndash Plasmid vector
ndash Binary vector a cloning vector used to generate transgenic plants
ndash Cloning vector
ndash Expression vector a plasmid specifically used for protein expression in thetarget
ndash Shuttle vector a vector (usually a plasmid) constructed so that it canpropagate in two different host species
ndash Viral vector a virus modified to deliver foreign genetic material into a cell
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Vectors can be classified based on
bull The purpose of the vector ndash eg cloning library construction ssDNA production mRNA synthesis expression
multipurpose etc
bull Origin of vector ndash Plasmid ndash Bacteriophage ndash Cosmid ndash Phagemid ndash YAC BAC
bull The host organism that the vector is propagated in ndash Bacteria (bacterial origin of replication) ndash Yeast (yeast origin of replication) ndash Shuttle vector (both origins)
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Types of vectors
Vector Target fragment length
Plasmid 0-10 kb (total size up to 15 kb)
Cosmid 10-40 kb
P1 artificial chromosome (PAC) 130-150 kbBacterial artificial chromosome (BAC) About 300 kb
Yeast artificial chromosome (YAC) 200 kb to 2 Mb
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What vectors will I be covering
Vectors
bull Plasmid vectorso Cloning vectors
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Plasmid Vectors
bull Plasmids most commonly used in recombinant DNA technology replicate in E coli
bull They have been engineered to optimize their use as vectors in DNA cloning
ndash To simplify working with plasmids their length is reduced
ndash Most plasmid vectors contain little more than the essential nucleotide
sequences required for their use in DNA cloning
bull Plasmid vectors are usually used as either cloning vectors expression vectorsssDNA vectors or transformation vectors
bull They are not used as library vectors
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Basic Characteristics of Cloning Vectors
Basic characteristics
1 Origin of replication (ori )
ndash An origin of replication is a sequence of DNA at which replication is initiated on achromosome plasmid or virus
bull small DNAs a single origin is sufficient ndash Larger DNAs have many origins and DNA replication is initiated at all of them
bull Determines the vector copy number
bull Determines the plasmids compatibility its ability to replicate in conjunction withanother plasmid within the same bacterial cell
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Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
8172019 Restriction Enzymes and Vectors
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Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
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Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
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Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
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Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
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Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
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Expression vectors
Bacterial Artificial Chromosome (BAC)
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Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
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Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
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A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
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Next DayTransformation SelectionScreening
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P
P P
P
DNA fragmentVector
tetRAmpR
DNA ligase
- Recombinant DNA- Recombinant vector- DNA chimera
Cut with restrictionendonuclease
8172019 Restriction Enzymes and Vectors
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Recombinant-DNA technology1 Restriction digestion
2 Joining DNA molecules3 Transformation
4 Selection Screening = identifying-
DNA molecule5 Cloning
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Restriction endonucleases
bull Also called restriction enzymes
bull Restriction endonucleases enzymes found in bacteria capable ofspecifically binding to DNA and cleaving dsDNA
bull Restriction endonucleases along with DNA Methylases(modifying enzymes) form a bacterial defense system againstbacteriophages
8172019 Restriction Enzymes and Vectors
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Restriction Endonucleases- defense function
bull Methyltransferases or methylases add a methyl group to DNA in
some bacteria
bull Restriction endonucleases will not cleave at their restriction siteif it is methylated
bull Invading DNA usually not methylated will be cleaved and thusinactivated by the bacteria whose own genome is methylated
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Restriction Endonucleases - three typestype I II and III
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Restriction Endonucleases
R-DNA technology uses type II ndash Why
1 Restriction activity and modifying activity are in separateenzymes
2 Restriction endonucleases cleave within the recognition site oradjacent to it
3 Do not need ATP for activity Needs only Mg++
4 Cuts in a precise manner very predictable
5 Short palindromic recognition sequence
Note
Over 1500 known over 150 available commercially
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Restriction Endonucleases- Nomenclature
Named for bacterial genus species strain and type
xamp e coGenus E scherichia Species co li Strain ROrder discovered 1
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Restriction Endonucleases- Nomenclature
eg Bam H1Bacillus amyloliquefaciens Strain H
First enzyme characterised
eg Hin dIIIHaemophilus influenzae Strain D - 3rd enzyme
eg Eco RI
Escherichia coli Strain R ndash 1st enzyme discovered
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Restriction Endonucleases
Recognition sites have symmetry (palindromic)
ldquoAble was I ere I saw Elbardquo
Bam H1 site5rsquo-GGATCC-3rsquo
3rsquo-CCTAGG-5rsquo
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Restriction Endonucleases
eg Bam H1
Cleavage site
GGATCCCCTAGG
Recognition sequence
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Restriction Endonucleases
Cohesive end cutter or sticky end cutter
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Restriction Endonucleases
eg Sma I = Serratia marcescens Enzyme 1
CCCGGG
Cleavage site
GGGCCC
Recognition sequence
BLUNT END CUTTEReg Sca1 EcoRV Nru1
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Classification of Restriction Endonucleases
1 Sticky-end cutter vs blunt-end cutter
2 Frequent cutter vs rare cutter
Recognition sequence Cleavage frequency
4 bp (Alu1 HpaII HhaI) (144) = 1256
5 bp (Hga1) (145 ) = 110246 bp (BamH1 HinD111) (146 ) = 14096
7 bp (PpuM1) (147 ) = 116384
8 bp (Not1)
9 bp (AlwN1)10 bp
8172019 Restriction Enzymes and Vectors
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983122983141983139983151983149983138983145983150983137983150983156 983140983150983137 983154983141983155983156983154983145983139983156983145983151983150
983141983150983140983151983150983157983139983148983141983137983155983141983155
8172019 Restriction Enzymes and Vectors
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Classification of Restriction Endonucleases
3 Ambiguous vs precise cutters
eg BsiE1
CGPuPyCGGCPyPuGC
8172019 Restriction Enzymes and Vectors
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Classification of Restriction Endonucleases
4 Isoshizomers
Some enzymes have the same target site (recognition site)
Two subtypes
Same target site ndash different cleavage site
eg Sma1 and Xma1
Same target site - same cleavage site
eg Aat1 and Stu1
BamH1 and Bst1
8172019 Restriction Enzymes and Vectors
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8172019 Restriction Enzymes and Vectors
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8172019 Restriction Enzymes and Vectors
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Restriction digestionCommercial restriction enzymes
1 Restriction enzymes are commercially available at 5 units per
microL concentration
2 I unit = 1 unit (U) is the amount if enzyme that catalyses the
conditions
3 Enzymes come in storage buffer containing EDTA DTT BSAand Glycerol
4 Too much enzyme in the reaction mix can cause star activity
8172019 Restriction Enzymes and Vectors
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Star activity
Loss of specificity of the restriction enzyme (typeII) underextreme non-standard conditions is called star activity
Under such conditions the restriction enzyme may cleave similarbut non identical target sequences
Non-standard conditions include1 high glycerol concentration gt5 vv
2 gt100 units per ug of DNA
3 Low ionic strength buffer lt25mM (usually is 100-150 mM)
4 high pH (gt 80) usually pH is around 75 presence of organic solvents DMSO ethanol PEGdimethyl acetamide
6 Cations other than Mg++ eg Cu++ Co++Zn++ Mn++
8172019 Restriction Enzymes and Vectors
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Restriction digestion
Stopping the Restriction digestion
1 Heat inactivation of restriction enzymes
Place reaction mix in a waterbath set at 65oCNote not all Restriction enzymes can be heat inactivated egPvu1 Bgl II BamH1 Acc1
2 Add 2ul of 25 mM EDTAEDTA chelates the Mg++ and makes it unavailable to theRestriction Endonuclease
3 Phenol-chloroform extraction purificationRemoves all proteins including the enzymes
8172019 Restriction Enzymes and Vectors
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EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Single Digests
EcoRI BamHI NcoI
Number offragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2753
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
U n d i g e s t e d
N c o I
EcoRI BamHI NcoI
Numberof
fragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2853
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Double Digests
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2953
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
E c o R I
N c o I
U n d i g e s t e d
B a m H I
N c o I
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3053
SummaryRestriction Digestion is the process of cutting DNA molecules into smaller pieces
with special enzymes called Restriction Endonucleases (sometimes just called
Restriction Enzymes or REs) These special enzymes recognize specificsequences in the DNA molecule (for example GATATC) wherever that sequenceoccurs in the DNA
httphigheredmcgraw-hillcomsites9834092339student_view0chapter18restriction_endonucleaseshtml
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Vectors
8172019 Restriction Enzymes and Vectors
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So what are vectors
bull Vector (molecular biology) vehicle used to transfer genetic material to a target
cell
8172019 Restriction Enzymes and Vectors
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So what are vectors
ndash Plasmid vector
ndash Binary vector a cloning vector used to generate transgenic plants
ndash Cloning vector
ndash Expression vector a plasmid specifically used for protein expression in thetarget
ndash Shuttle vector a vector (usually a plasmid) constructed so that it canpropagate in two different host species
ndash Viral vector a virus modified to deliver foreign genetic material into a cell
8172019 Restriction Enzymes and Vectors
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Vectors can be classified based on
bull The purpose of the vector ndash eg cloning library construction ssDNA production mRNA synthesis expression
multipurpose etc
bull Origin of vector ndash Plasmid ndash Bacteriophage ndash Cosmid ndash Phagemid ndash YAC BAC
bull The host organism that the vector is propagated in ndash Bacteria (bacterial origin of replication) ndash Yeast (yeast origin of replication) ndash Shuttle vector (both origins)
8172019 Restriction Enzymes and Vectors
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Types of vectors
Vector Target fragment length
Plasmid 0-10 kb (total size up to 15 kb)
Cosmid 10-40 kb
P1 artificial chromosome (PAC) 130-150 kbBacterial artificial chromosome (BAC) About 300 kb
Yeast artificial chromosome (YAC) 200 kb to 2 Mb
8172019 Restriction Enzymes and Vectors
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What vectors will I be covering
Vectors
bull Plasmid vectorso Cloning vectors
8172019 Restriction Enzymes and Vectors
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8172019 Restriction Enzymes and Vectors
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Plasmid Vectors
bull Plasmids most commonly used in recombinant DNA technology replicate in E coli
bull They have been engineered to optimize their use as vectors in DNA cloning
ndash To simplify working with plasmids their length is reduced
ndash Most plasmid vectors contain little more than the essential nucleotide
sequences required for their use in DNA cloning
bull Plasmid vectors are usually used as either cloning vectors expression vectorsssDNA vectors or transformation vectors
bull They are not used as library vectors
8172019 Restriction Enzymes and Vectors
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8172019 Restriction Enzymes and Vectors
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Basic Characteristics of Cloning Vectors
Basic characteristics
1 Origin of replication (ori )
ndash An origin of replication is a sequence of DNA at which replication is initiated on achromosome plasmid or virus
bull small DNAs a single origin is sufficient ndash Larger DNAs have many origins and DNA replication is initiated at all of them
bull Determines the vector copy number
bull Determines the plasmids compatibility its ability to replicate in conjunction withanother plasmid within the same bacterial cell
8172019 Restriction Enzymes and Vectors
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Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
8172019 Restriction Enzymes and Vectors
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Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
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Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
8172019 Restriction Enzymes and Vectors
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Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
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Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
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Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
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Expression vectors
Bacterial Artificial Chromosome (BAC)
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Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
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Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
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A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
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Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
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Recombinant-DNA technology1 Restriction digestion
2 Joining DNA molecules3 Transformation
4 Selection Screening = identifying-
DNA molecule5 Cloning
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Restriction endonucleases
bull Also called restriction enzymes
bull Restriction endonucleases enzymes found in bacteria capable ofspecifically binding to DNA and cleaving dsDNA
bull Restriction endonucleases along with DNA Methylases(modifying enzymes) form a bacterial defense system againstbacteriophages
8172019 Restriction Enzymes and Vectors
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Restriction Endonucleases- defense function
bull Methyltransferases or methylases add a methyl group to DNA in
some bacteria
bull Restriction endonucleases will not cleave at their restriction siteif it is methylated
bull Invading DNA usually not methylated will be cleaved and thusinactivated by the bacteria whose own genome is methylated
8172019 Restriction Enzymes and Vectors
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Restriction Endonucleases - three typestype I II and III
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Restriction Endonucleases
R-DNA technology uses type II ndash Why
1 Restriction activity and modifying activity are in separateenzymes
2 Restriction endonucleases cleave within the recognition site oradjacent to it
3 Do not need ATP for activity Needs only Mg++
4 Cuts in a precise manner very predictable
5 Short palindromic recognition sequence
Note
Over 1500 known over 150 available commercially
8172019 Restriction Enzymes and Vectors
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Restriction Endonucleases- Nomenclature
Named for bacterial genus species strain and type
xamp e coGenus E scherichia Species co li Strain ROrder discovered 1
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Restriction Endonucleases- Nomenclature
eg Bam H1Bacillus amyloliquefaciens Strain H
First enzyme characterised
eg Hin dIIIHaemophilus influenzae Strain D - 3rd enzyme
eg Eco RI
Escherichia coli Strain R ndash 1st enzyme discovered
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Restriction Endonucleases
Recognition sites have symmetry (palindromic)
ldquoAble was I ere I saw Elbardquo
Bam H1 site5rsquo-GGATCC-3rsquo
3rsquo-CCTAGG-5rsquo
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Restriction Endonucleases
eg Bam H1
Cleavage site
GGATCCCCTAGG
Recognition sequence
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Restriction Endonucleases
Cohesive end cutter or sticky end cutter
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Restriction Endonucleases
eg Sma I = Serratia marcescens Enzyme 1
CCCGGG
Cleavage site
GGGCCC
Recognition sequence
BLUNT END CUTTEReg Sca1 EcoRV Nru1
8172019 Restriction Enzymes and Vectors
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Classification of Restriction Endonucleases
1 Sticky-end cutter vs blunt-end cutter
2 Frequent cutter vs rare cutter
Recognition sequence Cleavage frequency
4 bp (Alu1 HpaII HhaI) (144) = 1256
5 bp (Hga1) (145 ) = 110246 bp (BamH1 HinD111) (146 ) = 14096
7 bp (PpuM1) (147 ) = 116384
8 bp (Not1)
9 bp (AlwN1)10 bp
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983122983141983139983151983149983138983145983150983137983150983156 983140983150983137 983154983141983155983156983154983145983139983156983145983151983150
983141983150983140983151983150983157983139983148983141983137983155983141983155
8172019 Restriction Enzymes and Vectors
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Classification of Restriction Endonucleases
3 Ambiguous vs precise cutters
eg BsiE1
CGPuPyCGGCPyPuGC
8172019 Restriction Enzymes and Vectors
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Classification of Restriction Endonucleases
4 Isoshizomers
Some enzymes have the same target site (recognition site)
Two subtypes
Same target site ndash different cleavage site
eg Sma1 and Xma1
Same target site - same cleavage site
eg Aat1 and Stu1
BamH1 and Bst1
8172019 Restriction Enzymes and Vectors
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Restriction digestionCommercial restriction enzymes
1 Restriction enzymes are commercially available at 5 units per
microL concentration
2 I unit = 1 unit (U) is the amount if enzyme that catalyses the
conditions
3 Enzymes come in storage buffer containing EDTA DTT BSAand Glycerol
4 Too much enzyme in the reaction mix can cause star activity
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2453
Star activity
Loss of specificity of the restriction enzyme (typeII) underextreme non-standard conditions is called star activity
Under such conditions the restriction enzyme may cleave similarbut non identical target sequences
Non-standard conditions include1 high glycerol concentration gt5 vv
2 gt100 units per ug of DNA
3 Low ionic strength buffer lt25mM (usually is 100-150 mM)
4 high pH (gt 80) usually pH is around 75 presence of organic solvents DMSO ethanol PEGdimethyl acetamide
6 Cations other than Mg++ eg Cu++ Co++Zn++ Mn++
8172019 Restriction Enzymes and Vectors
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Restriction digestion
Stopping the Restriction digestion
1 Heat inactivation of restriction enzymes
Place reaction mix in a waterbath set at 65oCNote not all Restriction enzymes can be heat inactivated egPvu1 Bgl II BamH1 Acc1
2 Add 2ul of 25 mM EDTAEDTA chelates the Mg++ and makes it unavailable to theRestriction Endonuclease
3 Phenol-chloroform extraction purificationRemoves all proteins including the enzymes
8172019 Restriction Enzymes and Vectors
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EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Single Digests
EcoRI BamHI NcoI
Number offragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2753
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
U n d i g e s t e d
N c o I
EcoRI BamHI NcoI
Numberof
fragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2853
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Double Digests
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2953
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
E c o R I
N c o I
U n d i g e s t e d
B a m H I
N c o I
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
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SummaryRestriction Digestion is the process of cutting DNA molecules into smaller pieces
with special enzymes called Restriction Endonucleases (sometimes just called
Restriction Enzymes or REs) These special enzymes recognize specificsequences in the DNA molecule (for example GATATC) wherever that sequenceoccurs in the DNA
httphigheredmcgraw-hillcomsites9834092339student_view0chapter18restriction_endonucleaseshtml
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Vectors
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So what are vectors
bull Vector (molecular biology) vehicle used to transfer genetic material to a target
cell
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So what are vectors
ndash Plasmid vector
ndash Binary vector a cloning vector used to generate transgenic plants
ndash Cloning vector
ndash Expression vector a plasmid specifically used for protein expression in thetarget
ndash Shuttle vector a vector (usually a plasmid) constructed so that it canpropagate in two different host species
ndash Viral vector a virus modified to deliver foreign genetic material into a cell
8172019 Restriction Enzymes and Vectors
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Vectors can be classified based on
bull The purpose of the vector ndash eg cloning library construction ssDNA production mRNA synthesis expression
multipurpose etc
bull Origin of vector ndash Plasmid ndash Bacteriophage ndash Cosmid ndash Phagemid ndash YAC BAC
bull The host organism that the vector is propagated in ndash Bacteria (bacterial origin of replication) ndash Yeast (yeast origin of replication) ndash Shuttle vector (both origins)
8172019 Restriction Enzymes and Vectors
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Types of vectors
Vector Target fragment length
Plasmid 0-10 kb (total size up to 15 kb)
Cosmid 10-40 kb
P1 artificial chromosome (PAC) 130-150 kbBacterial artificial chromosome (BAC) About 300 kb
Yeast artificial chromosome (YAC) 200 kb to 2 Mb
8172019 Restriction Enzymes and Vectors
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What vectors will I be covering
Vectors
bull Plasmid vectorso Cloning vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3753
8172019 Restriction Enzymes and Vectors
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Plasmid Vectors
bull Plasmids most commonly used in recombinant DNA technology replicate in E coli
bull They have been engineered to optimize their use as vectors in DNA cloning
ndash To simplify working with plasmids their length is reduced
ndash Most plasmid vectors contain little more than the essential nucleotide
sequences required for their use in DNA cloning
bull Plasmid vectors are usually used as either cloning vectors expression vectorsssDNA vectors or transformation vectors
bull They are not used as library vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3953
8172019 Restriction Enzymes and Vectors
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8172019 Restriction Enzymes and Vectors
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Basic Characteristics of Cloning Vectors
Basic characteristics
1 Origin of replication (ori )
ndash An origin of replication is a sequence of DNA at which replication is initiated on achromosome plasmid or virus
bull small DNAs a single origin is sufficient ndash Larger DNAs have many origins and DNA replication is initiated at all of them
bull Determines the vector copy number
bull Determines the plasmids compatibility its ability to replicate in conjunction withanother plasmid within the same bacterial cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4353
Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4453
Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
8172019 Restriction Enzymes and Vectors
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Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
8172019 Restriction Enzymes and Vectors
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Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4753
Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
8172019 Restriction Enzymes and Vectors
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Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
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Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
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Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
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Next DayTransformation SelectionScreening
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Restriction endonucleases
bull Also called restriction enzymes
bull Restriction endonucleases enzymes found in bacteria capable ofspecifically binding to DNA and cleaving dsDNA
bull Restriction endonucleases along with DNA Methylases(modifying enzymes) form a bacterial defense system againstbacteriophages
8172019 Restriction Enzymes and Vectors
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Restriction Endonucleases- defense function
bull Methyltransferases or methylases add a methyl group to DNA in
some bacteria
bull Restriction endonucleases will not cleave at their restriction siteif it is methylated
bull Invading DNA usually not methylated will be cleaved and thusinactivated by the bacteria whose own genome is methylated
8172019 Restriction Enzymes and Vectors
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Restriction Endonucleases - three typestype I II and III
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Restriction Endonucleases
R-DNA technology uses type II ndash Why
1 Restriction activity and modifying activity are in separateenzymes
2 Restriction endonucleases cleave within the recognition site oradjacent to it
3 Do not need ATP for activity Needs only Mg++
4 Cuts in a precise manner very predictable
5 Short palindromic recognition sequence
Note
Over 1500 known over 150 available commercially
8172019 Restriction Enzymes and Vectors
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Restriction Endonucleases- Nomenclature
Named for bacterial genus species strain and type
xamp e coGenus E scherichia Species co li Strain ROrder discovered 1
8172019 Restriction Enzymes and Vectors
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Restriction Endonucleases- Nomenclature
eg Bam H1Bacillus amyloliquefaciens Strain H
First enzyme characterised
eg Hin dIIIHaemophilus influenzae Strain D - 3rd enzyme
eg Eco RI
Escherichia coli Strain R ndash 1st enzyme discovered
8172019 Restriction Enzymes and Vectors
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Restriction Endonucleases
Recognition sites have symmetry (palindromic)
ldquoAble was I ere I saw Elbardquo
Bam H1 site5rsquo-GGATCC-3rsquo
3rsquo-CCTAGG-5rsquo
8172019 Restriction Enzymes and Vectors
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Restriction Endonucleases
eg Bam H1
Cleavage site
GGATCCCCTAGG
Recognition sequence
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Restriction Endonucleases
Cohesive end cutter or sticky end cutter
8172019 Restriction Enzymes and Vectors
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Restriction Endonucleases
eg Sma I = Serratia marcescens Enzyme 1
CCCGGG
Cleavage site
GGGCCC
Recognition sequence
BLUNT END CUTTEReg Sca1 EcoRV Nru1
8172019 Restriction Enzymes and Vectors
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Classification of Restriction Endonucleases
1 Sticky-end cutter vs blunt-end cutter
2 Frequent cutter vs rare cutter
Recognition sequence Cleavage frequency
4 bp (Alu1 HpaII HhaI) (144) = 1256
5 bp (Hga1) (145 ) = 110246 bp (BamH1 HinD111) (146 ) = 14096
7 bp (PpuM1) (147 ) = 116384
8 bp (Not1)
9 bp (AlwN1)10 bp
8172019 Restriction Enzymes and Vectors
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983122983141983139983151983149983138983145983150983137983150983156 983140983150983137 983154983141983155983156983154983145983139983156983145983151983150
983141983150983140983151983150983157983139983148983141983137983155983141983155
8172019 Restriction Enzymes and Vectors
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Classification of Restriction Endonucleases
3 Ambiguous vs precise cutters
eg BsiE1
CGPuPyCGGCPyPuGC
8172019 Restriction Enzymes and Vectors
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Classification of Restriction Endonucleases
4 Isoshizomers
Some enzymes have the same target site (recognition site)
Two subtypes
Same target site ndash different cleavage site
eg Sma1 and Xma1
Same target site - same cleavage site
eg Aat1 and Stu1
BamH1 and Bst1
8172019 Restriction Enzymes and Vectors
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Restriction digestionCommercial restriction enzymes
1 Restriction enzymes are commercially available at 5 units per
microL concentration
2 I unit = 1 unit (U) is the amount if enzyme that catalyses the
conditions
3 Enzymes come in storage buffer containing EDTA DTT BSAand Glycerol
4 Too much enzyme in the reaction mix can cause star activity
8172019 Restriction Enzymes and Vectors
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Star activity
Loss of specificity of the restriction enzyme (typeII) underextreme non-standard conditions is called star activity
Under such conditions the restriction enzyme may cleave similarbut non identical target sequences
Non-standard conditions include1 high glycerol concentration gt5 vv
2 gt100 units per ug of DNA
3 Low ionic strength buffer lt25mM (usually is 100-150 mM)
4 high pH (gt 80) usually pH is around 75 presence of organic solvents DMSO ethanol PEGdimethyl acetamide
6 Cations other than Mg++ eg Cu++ Co++Zn++ Mn++
8172019 Restriction Enzymes and Vectors
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Restriction digestion
Stopping the Restriction digestion
1 Heat inactivation of restriction enzymes
Place reaction mix in a waterbath set at 65oCNote not all Restriction enzymes can be heat inactivated egPvu1 Bgl II BamH1 Acc1
2 Add 2ul of 25 mM EDTAEDTA chelates the Mg++ and makes it unavailable to theRestriction Endonuclease
3 Phenol-chloroform extraction purificationRemoves all proteins including the enzymes
8172019 Restriction Enzymes and Vectors
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EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Single Digests
EcoRI BamHI NcoI
Number offragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
8172019 Restriction Enzymes and Vectors
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Digestions
4kbp
L a d d e r
E c o R I
B a m H I
U n d i g e s t e d
N c o I
EcoRI BamHI NcoI
Numberof
fragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
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EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Double Digests
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2953
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
E c o R I
N c o I
U n d i g e s t e d
B a m H I
N c o I
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
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SummaryRestriction Digestion is the process of cutting DNA molecules into smaller pieces
with special enzymes called Restriction Endonucleases (sometimes just called
Restriction Enzymes or REs) These special enzymes recognize specificsequences in the DNA molecule (for example GATATC) wherever that sequenceoccurs in the DNA
httphigheredmcgraw-hillcomsites9834092339student_view0chapter18restriction_endonucleaseshtml
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Vectors
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So what are vectors
bull Vector (molecular biology) vehicle used to transfer genetic material to a target
cell
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So what are vectors
ndash Plasmid vector
ndash Binary vector a cloning vector used to generate transgenic plants
ndash Cloning vector
ndash Expression vector a plasmid specifically used for protein expression in thetarget
ndash Shuttle vector a vector (usually a plasmid) constructed so that it canpropagate in two different host species
ndash Viral vector a virus modified to deliver foreign genetic material into a cell
8172019 Restriction Enzymes and Vectors
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Vectors can be classified based on
bull The purpose of the vector ndash eg cloning library construction ssDNA production mRNA synthesis expression
multipurpose etc
bull Origin of vector ndash Plasmid ndash Bacteriophage ndash Cosmid ndash Phagemid ndash YAC BAC
bull The host organism that the vector is propagated in ndash Bacteria (bacterial origin of replication) ndash Yeast (yeast origin of replication) ndash Shuttle vector (both origins)
8172019 Restriction Enzymes and Vectors
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Types of vectors
Vector Target fragment length
Plasmid 0-10 kb (total size up to 15 kb)
Cosmid 10-40 kb
P1 artificial chromosome (PAC) 130-150 kbBacterial artificial chromosome (BAC) About 300 kb
Yeast artificial chromosome (YAC) 200 kb to 2 Mb
8172019 Restriction Enzymes and Vectors
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What vectors will I be covering
Vectors
bull Plasmid vectorso Cloning vectors
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Plasmid Vectors
bull Plasmids most commonly used in recombinant DNA technology replicate in E coli
bull They have been engineered to optimize their use as vectors in DNA cloning
ndash To simplify working with plasmids their length is reduced
ndash Most plasmid vectors contain little more than the essential nucleotide
sequences required for their use in DNA cloning
bull Plasmid vectors are usually used as either cloning vectors expression vectorsssDNA vectors or transformation vectors
bull They are not used as library vectors
8172019 Restriction Enzymes and Vectors
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Basic Characteristics of Cloning Vectors
Basic characteristics
1 Origin of replication (ori )
ndash An origin of replication is a sequence of DNA at which replication is initiated on achromosome plasmid or virus
bull small DNAs a single origin is sufficient ndash Larger DNAs have many origins and DNA replication is initiated at all of them
bull Determines the vector copy number
bull Determines the plasmids compatibility its ability to replicate in conjunction withanother plasmid within the same bacterial cell
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Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
8172019 Restriction Enzymes and Vectors
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Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
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Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
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Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
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Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
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Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
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Expression vectors
Bacterial Artificial Chromosome (BAC)
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Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
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Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
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A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
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Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
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Restriction Endonucleases- defense function
bull Methyltransferases or methylases add a methyl group to DNA in
some bacteria
bull Restriction endonucleases will not cleave at their restriction siteif it is methylated
bull Invading DNA usually not methylated will be cleaved and thusinactivated by the bacteria whose own genome is methylated
8172019 Restriction Enzymes and Vectors
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8172019 Restriction Enzymes and Vectors
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Restriction Endonucleases - three typestype I II and III
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Restriction Endonucleases
R-DNA technology uses type II ndash Why
1 Restriction activity and modifying activity are in separateenzymes
2 Restriction endonucleases cleave within the recognition site oradjacent to it
3 Do not need ATP for activity Needs only Mg++
4 Cuts in a precise manner very predictable
5 Short palindromic recognition sequence
Note
Over 1500 known over 150 available commercially
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1153
Restriction Endonucleases- Nomenclature
Named for bacterial genus species strain and type
xamp e coGenus E scherichia Species co li Strain ROrder discovered 1
8172019 Restriction Enzymes and Vectors
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Restriction Endonucleases- Nomenclature
eg Bam H1Bacillus amyloliquefaciens Strain H
First enzyme characterised
eg Hin dIIIHaemophilus influenzae Strain D - 3rd enzyme
eg Eco RI
Escherichia coli Strain R ndash 1st enzyme discovered
8172019 Restriction Enzymes and Vectors
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Restriction Endonucleases
Recognition sites have symmetry (palindromic)
ldquoAble was I ere I saw Elbardquo
Bam H1 site5rsquo-GGATCC-3rsquo
3rsquo-CCTAGG-5rsquo
8172019 Restriction Enzymes and Vectors
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Restriction Endonucleases
eg Bam H1
Cleavage site
GGATCCCCTAGG
Recognition sequence
8172019 Restriction Enzymes and Vectors
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Restriction Endonucleases
Cohesive end cutter or sticky end cutter
8172019 Restriction Enzymes and Vectors
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Restriction Endonucleases
eg Sma I = Serratia marcescens Enzyme 1
CCCGGG
Cleavage site
GGGCCC
Recognition sequence
BLUNT END CUTTEReg Sca1 EcoRV Nru1
8172019 Restriction Enzymes and Vectors
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Classification of Restriction Endonucleases
1 Sticky-end cutter vs blunt-end cutter
2 Frequent cutter vs rare cutter
Recognition sequence Cleavage frequency
4 bp (Alu1 HpaII HhaI) (144) = 1256
5 bp (Hga1) (145 ) = 110246 bp (BamH1 HinD111) (146 ) = 14096
7 bp (PpuM1) (147 ) = 116384
8 bp (Not1)
9 bp (AlwN1)10 bp
8172019 Restriction Enzymes and Vectors
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983122983141983139983151983149983138983145983150983137983150983156 983140983150983137 983154983141983155983156983154983145983139983156983145983151983150
983141983150983140983151983150983157983139983148983141983137983155983141983155
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1953
Classification of Restriction Endonucleases
3 Ambiguous vs precise cutters
eg BsiE1
CGPuPyCGGCPyPuGC
8172019 Restriction Enzymes and Vectors
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Classification of Restriction Endonucleases
4 Isoshizomers
Some enzymes have the same target site (recognition site)
Two subtypes
Same target site ndash different cleavage site
eg Sma1 and Xma1
Same target site - same cleavage site
eg Aat1 and Stu1
BamH1 and Bst1
8172019 Restriction Enzymes and Vectors
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8172019 Restriction Enzymes and Vectors
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8172019 Restriction Enzymes and Vectors
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Restriction digestionCommercial restriction enzymes
1 Restriction enzymes are commercially available at 5 units per
microL concentration
2 I unit = 1 unit (U) is the amount if enzyme that catalyses the
conditions
3 Enzymes come in storage buffer containing EDTA DTT BSAand Glycerol
4 Too much enzyme in the reaction mix can cause star activity
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2453
Star activity
Loss of specificity of the restriction enzyme (typeII) underextreme non-standard conditions is called star activity
Under such conditions the restriction enzyme may cleave similarbut non identical target sequences
Non-standard conditions include1 high glycerol concentration gt5 vv
2 gt100 units per ug of DNA
3 Low ionic strength buffer lt25mM (usually is 100-150 mM)
4 high pH (gt 80) usually pH is around 75 presence of organic solvents DMSO ethanol PEGdimethyl acetamide
6 Cations other than Mg++ eg Cu++ Co++Zn++ Mn++
8172019 Restriction Enzymes and Vectors
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Restriction digestion
Stopping the Restriction digestion
1 Heat inactivation of restriction enzymes
Place reaction mix in a waterbath set at 65oCNote not all Restriction enzymes can be heat inactivated egPvu1 Bgl II BamH1 Acc1
2 Add 2ul of 25 mM EDTAEDTA chelates the Mg++ and makes it unavailable to theRestriction Endonuclease
3 Phenol-chloroform extraction purificationRemoves all proteins including the enzymes
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2653
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Single Digests
EcoRI BamHI NcoI
Number offragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2753
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
U n d i g e s t e d
N c o I
EcoRI BamHI NcoI
Numberof
fragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2853
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Double Digests
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2953
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
E c o R I
N c o I
U n d i g e s t e d
B a m H I
N c o I
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3053
SummaryRestriction Digestion is the process of cutting DNA molecules into smaller pieces
with special enzymes called Restriction Endonucleases (sometimes just called
Restriction Enzymes or REs) These special enzymes recognize specificsequences in the DNA molecule (for example GATATC) wherever that sequenceoccurs in the DNA
httphigheredmcgraw-hillcomsites9834092339student_view0chapter18restriction_endonucleaseshtml
8172019 Restriction Enzymes and Vectors
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Vectors
8172019 Restriction Enzymes and Vectors
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So what are vectors
bull Vector (molecular biology) vehicle used to transfer genetic material to a target
cell
8172019 Restriction Enzymes and Vectors
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So what are vectors
ndash Plasmid vector
ndash Binary vector a cloning vector used to generate transgenic plants
ndash Cloning vector
ndash Expression vector a plasmid specifically used for protein expression in thetarget
ndash Shuttle vector a vector (usually a plasmid) constructed so that it canpropagate in two different host species
ndash Viral vector a virus modified to deliver foreign genetic material into a cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3453
Vectors can be classified based on
bull The purpose of the vector ndash eg cloning library construction ssDNA production mRNA synthesis expression
multipurpose etc
bull Origin of vector ndash Plasmid ndash Bacteriophage ndash Cosmid ndash Phagemid ndash YAC BAC
bull The host organism that the vector is propagated in ndash Bacteria (bacterial origin of replication) ndash Yeast (yeast origin of replication) ndash Shuttle vector (both origins)
8172019 Restriction Enzymes and Vectors
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Types of vectors
Vector Target fragment length
Plasmid 0-10 kb (total size up to 15 kb)
Cosmid 10-40 kb
P1 artificial chromosome (PAC) 130-150 kbBacterial artificial chromosome (BAC) About 300 kb
Yeast artificial chromosome (YAC) 200 kb to 2 Mb
8172019 Restriction Enzymes and Vectors
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What vectors will I be covering
Vectors
bull Plasmid vectorso Cloning vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3753
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3853
Plasmid Vectors
bull Plasmids most commonly used in recombinant DNA technology replicate in E coli
bull They have been engineered to optimize their use as vectors in DNA cloning
ndash To simplify working with plasmids their length is reduced
ndash Most plasmid vectors contain little more than the essential nucleotide
sequences required for their use in DNA cloning
bull Plasmid vectors are usually used as either cloning vectors expression vectorsssDNA vectors or transformation vectors
bull They are not used as library vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3953
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4053
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4253
Basic Characteristics of Cloning Vectors
Basic characteristics
1 Origin of replication (ori )
ndash An origin of replication is a sequence of DNA at which replication is initiated on achromosome plasmid or virus
bull small DNAs a single origin is sufficient ndash Larger DNAs have many origins and DNA replication is initiated at all of them
bull Determines the vector copy number
bull Determines the plasmids compatibility its ability to replicate in conjunction withanother plasmid within the same bacterial cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4353
Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4453
Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4553
Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4653
Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4753
Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4853
Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 853
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 953
Restriction Endonucleases - three typestype I II and III
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1053
Restriction Endonucleases
R-DNA technology uses type II ndash Why
1 Restriction activity and modifying activity are in separateenzymes
2 Restriction endonucleases cleave within the recognition site oradjacent to it
3 Do not need ATP for activity Needs only Mg++
4 Cuts in a precise manner very predictable
5 Short palindromic recognition sequence
Note
Over 1500 known over 150 available commercially
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1153
Restriction Endonucleases- Nomenclature
Named for bacterial genus species strain and type
xamp e coGenus E scherichia Species co li Strain ROrder discovered 1
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1253
Restriction Endonucleases- Nomenclature
eg Bam H1Bacillus amyloliquefaciens Strain H
First enzyme characterised
eg Hin dIIIHaemophilus influenzae Strain D - 3rd enzyme
eg Eco RI
Escherichia coli Strain R ndash 1st enzyme discovered
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1353
Restriction Endonucleases
Recognition sites have symmetry (palindromic)
ldquoAble was I ere I saw Elbardquo
Bam H1 site5rsquo-GGATCC-3rsquo
3rsquo-CCTAGG-5rsquo
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1453
Restriction Endonucleases
eg Bam H1
Cleavage site
GGATCCCCTAGG
Recognition sequence
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1553
Restriction Endonucleases
Cohesive end cutter or sticky end cutter
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1653
Restriction Endonucleases
eg Sma I = Serratia marcescens Enzyme 1
CCCGGG
Cleavage site
GGGCCC
Recognition sequence
BLUNT END CUTTEReg Sca1 EcoRV Nru1
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1753
Classification of Restriction Endonucleases
1 Sticky-end cutter vs blunt-end cutter
2 Frequent cutter vs rare cutter
Recognition sequence Cleavage frequency
4 bp (Alu1 HpaII HhaI) (144) = 1256
5 bp (Hga1) (145 ) = 110246 bp (BamH1 HinD111) (146 ) = 14096
7 bp (PpuM1) (147 ) = 116384
8 bp (Not1)
9 bp (AlwN1)10 bp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1853
983122983141983139983151983149983138983145983150983137983150983156 983140983150983137 983154983141983155983156983154983145983139983156983145983151983150
983141983150983140983151983150983157983139983148983141983137983155983141983155
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1953
Classification of Restriction Endonucleases
3 Ambiguous vs precise cutters
eg BsiE1
CGPuPyCGGCPyPuGC
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2053
Classification of Restriction Endonucleases
4 Isoshizomers
Some enzymes have the same target site (recognition site)
Two subtypes
Same target site ndash different cleavage site
eg Sma1 and Xma1
Same target site - same cleavage site
eg Aat1 and Stu1
BamH1 and Bst1
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2253
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2353
Restriction digestionCommercial restriction enzymes
1 Restriction enzymes are commercially available at 5 units per
microL concentration
2 I unit = 1 unit (U) is the amount if enzyme that catalyses the
conditions
3 Enzymes come in storage buffer containing EDTA DTT BSAand Glycerol
4 Too much enzyme in the reaction mix can cause star activity
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2453
Star activity
Loss of specificity of the restriction enzyme (typeII) underextreme non-standard conditions is called star activity
Under such conditions the restriction enzyme may cleave similarbut non identical target sequences
Non-standard conditions include1 high glycerol concentration gt5 vv
2 gt100 units per ug of DNA
3 Low ionic strength buffer lt25mM (usually is 100-150 mM)
4 high pH (gt 80) usually pH is around 75 presence of organic solvents DMSO ethanol PEGdimethyl acetamide
6 Cations other than Mg++ eg Cu++ Co++Zn++ Mn++
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2553
Restriction digestion
Stopping the Restriction digestion
1 Heat inactivation of restriction enzymes
Place reaction mix in a waterbath set at 65oCNote not all Restriction enzymes can be heat inactivated egPvu1 Bgl II BamH1 Acc1
2 Add 2ul of 25 mM EDTAEDTA chelates the Mg++ and makes it unavailable to theRestriction Endonuclease
3 Phenol-chloroform extraction purificationRemoves all proteins including the enzymes
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2653
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Single Digests
EcoRI BamHI NcoI
Number offragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2753
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
U n d i g e s t e d
N c o I
EcoRI BamHI NcoI
Numberof
fragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2853
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Double Digests
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2953
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
E c o R I
N c o I
U n d i g e s t e d
B a m H I
N c o I
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3053
SummaryRestriction Digestion is the process of cutting DNA molecules into smaller pieces
with special enzymes called Restriction Endonucleases (sometimes just called
Restriction Enzymes or REs) These special enzymes recognize specificsequences in the DNA molecule (for example GATATC) wherever that sequenceoccurs in the DNA
httphigheredmcgraw-hillcomsites9834092339student_view0chapter18restriction_endonucleaseshtml
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3153
Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3253
So what are vectors
bull Vector (molecular biology) vehicle used to transfer genetic material to a target
cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3353
So what are vectors
ndash Plasmid vector
ndash Binary vector a cloning vector used to generate transgenic plants
ndash Cloning vector
ndash Expression vector a plasmid specifically used for protein expression in thetarget
ndash Shuttle vector a vector (usually a plasmid) constructed so that it canpropagate in two different host species
ndash Viral vector a virus modified to deliver foreign genetic material into a cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3453
Vectors can be classified based on
bull The purpose of the vector ndash eg cloning library construction ssDNA production mRNA synthesis expression
multipurpose etc
bull Origin of vector ndash Plasmid ndash Bacteriophage ndash Cosmid ndash Phagemid ndash YAC BAC
bull The host organism that the vector is propagated in ndash Bacteria (bacterial origin of replication) ndash Yeast (yeast origin of replication) ndash Shuttle vector (both origins)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3553
Types of vectors
Vector Target fragment length
Plasmid 0-10 kb (total size up to 15 kb)
Cosmid 10-40 kb
P1 artificial chromosome (PAC) 130-150 kbBacterial artificial chromosome (BAC) About 300 kb
Yeast artificial chromosome (YAC) 200 kb to 2 Mb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3653
What vectors will I be covering
Vectors
bull Plasmid vectorso Cloning vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3753
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3853
Plasmid Vectors
bull Plasmids most commonly used in recombinant DNA technology replicate in E coli
bull They have been engineered to optimize their use as vectors in DNA cloning
ndash To simplify working with plasmids their length is reduced
ndash Most plasmid vectors contain little more than the essential nucleotide
sequences required for their use in DNA cloning
bull Plasmid vectors are usually used as either cloning vectors expression vectorsssDNA vectors or transformation vectors
bull They are not used as library vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3953
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4053
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4253
Basic Characteristics of Cloning Vectors
Basic characteristics
1 Origin of replication (ori )
ndash An origin of replication is a sequence of DNA at which replication is initiated on achromosome plasmid or virus
bull small DNAs a single origin is sufficient ndash Larger DNAs have many origins and DNA replication is initiated at all of them
bull Determines the vector copy number
bull Determines the plasmids compatibility its ability to replicate in conjunction withanother plasmid within the same bacterial cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4353
Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4453
Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4553
Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4653
Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4753
Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4853
Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 953
Restriction Endonucleases - three typestype I II and III
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1053
Restriction Endonucleases
R-DNA technology uses type II ndash Why
1 Restriction activity and modifying activity are in separateenzymes
2 Restriction endonucleases cleave within the recognition site oradjacent to it
3 Do not need ATP for activity Needs only Mg++
4 Cuts in a precise manner very predictable
5 Short palindromic recognition sequence
Note
Over 1500 known over 150 available commercially
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1153
Restriction Endonucleases- Nomenclature
Named for bacterial genus species strain and type
xamp e coGenus E scherichia Species co li Strain ROrder discovered 1
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1253
Restriction Endonucleases- Nomenclature
eg Bam H1Bacillus amyloliquefaciens Strain H
First enzyme characterised
eg Hin dIIIHaemophilus influenzae Strain D - 3rd enzyme
eg Eco RI
Escherichia coli Strain R ndash 1st enzyme discovered
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1353
Restriction Endonucleases
Recognition sites have symmetry (palindromic)
ldquoAble was I ere I saw Elbardquo
Bam H1 site5rsquo-GGATCC-3rsquo
3rsquo-CCTAGG-5rsquo
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1453
Restriction Endonucleases
eg Bam H1
Cleavage site
GGATCCCCTAGG
Recognition sequence
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1553
Restriction Endonucleases
Cohesive end cutter or sticky end cutter
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1653
Restriction Endonucleases
eg Sma I = Serratia marcescens Enzyme 1
CCCGGG
Cleavage site
GGGCCC
Recognition sequence
BLUNT END CUTTEReg Sca1 EcoRV Nru1
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1753
Classification of Restriction Endonucleases
1 Sticky-end cutter vs blunt-end cutter
2 Frequent cutter vs rare cutter
Recognition sequence Cleavage frequency
4 bp (Alu1 HpaII HhaI) (144) = 1256
5 bp (Hga1) (145 ) = 110246 bp (BamH1 HinD111) (146 ) = 14096
7 bp (PpuM1) (147 ) = 116384
8 bp (Not1)
9 bp (AlwN1)10 bp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1853
983122983141983139983151983149983138983145983150983137983150983156 983140983150983137 983154983141983155983156983154983145983139983156983145983151983150
983141983150983140983151983150983157983139983148983141983137983155983141983155
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1953
Classification of Restriction Endonucleases
3 Ambiguous vs precise cutters
eg BsiE1
CGPuPyCGGCPyPuGC
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2053
Classification of Restriction Endonucleases
4 Isoshizomers
Some enzymes have the same target site (recognition site)
Two subtypes
Same target site ndash different cleavage site
eg Sma1 and Xma1
Same target site - same cleavage site
eg Aat1 and Stu1
BamH1 and Bst1
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2253
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2353
Restriction digestionCommercial restriction enzymes
1 Restriction enzymes are commercially available at 5 units per
microL concentration
2 I unit = 1 unit (U) is the amount if enzyme that catalyses the
conditions
3 Enzymes come in storage buffer containing EDTA DTT BSAand Glycerol
4 Too much enzyme in the reaction mix can cause star activity
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2453
Star activity
Loss of specificity of the restriction enzyme (typeII) underextreme non-standard conditions is called star activity
Under such conditions the restriction enzyme may cleave similarbut non identical target sequences
Non-standard conditions include1 high glycerol concentration gt5 vv
2 gt100 units per ug of DNA
3 Low ionic strength buffer lt25mM (usually is 100-150 mM)
4 high pH (gt 80) usually pH is around 75 presence of organic solvents DMSO ethanol PEGdimethyl acetamide
6 Cations other than Mg++ eg Cu++ Co++Zn++ Mn++
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2553
Restriction digestion
Stopping the Restriction digestion
1 Heat inactivation of restriction enzymes
Place reaction mix in a waterbath set at 65oCNote not all Restriction enzymes can be heat inactivated egPvu1 Bgl II BamH1 Acc1
2 Add 2ul of 25 mM EDTAEDTA chelates the Mg++ and makes it unavailable to theRestriction Endonuclease
3 Phenol-chloroform extraction purificationRemoves all proteins including the enzymes
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2653
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Single Digests
EcoRI BamHI NcoI
Number offragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2753
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
U n d i g e s t e d
N c o I
EcoRI BamHI NcoI
Numberof
fragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2853
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Double Digests
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2953
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
E c o R I
N c o I
U n d i g e s t e d
B a m H I
N c o I
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3053
SummaryRestriction Digestion is the process of cutting DNA molecules into smaller pieces
with special enzymes called Restriction Endonucleases (sometimes just called
Restriction Enzymes or REs) These special enzymes recognize specificsequences in the DNA molecule (for example GATATC) wherever that sequenceoccurs in the DNA
httphigheredmcgraw-hillcomsites9834092339student_view0chapter18restriction_endonucleaseshtml
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3153
Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3253
So what are vectors
bull Vector (molecular biology) vehicle used to transfer genetic material to a target
cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3353
So what are vectors
ndash Plasmid vector
ndash Binary vector a cloning vector used to generate transgenic plants
ndash Cloning vector
ndash Expression vector a plasmid specifically used for protein expression in thetarget
ndash Shuttle vector a vector (usually a plasmid) constructed so that it canpropagate in two different host species
ndash Viral vector a virus modified to deliver foreign genetic material into a cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3453
Vectors can be classified based on
bull The purpose of the vector ndash eg cloning library construction ssDNA production mRNA synthesis expression
multipurpose etc
bull Origin of vector ndash Plasmid ndash Bacteriophage ndash Cosmid ndash Phagemid ndash YAC BAC
bull The host organism that the vector is propagated in ndash Bacteria (bacterial origin of replication) ndash Yeast (yeast origin of replication) ndash Shuttle vector (both origins)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3553
Types of vectors
Vector Target fragment length
Plasmid 0-10 kb (total size up to 15 kb)
Cosmid 10-40 kb
P1 artificial chromosome (PAC) 130-150 kbBacterial artificial chromosome (BAC) About 300 kb
Yeast artificial chromosome (YAC) 200 kb to 2 Mb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3653
What vectors will I be covering
Vectors
bull Plasmid vectorso Cloning vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3753
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3853
Plasmid Vectors
bull Plasmids most commonly used in recombinant DNA technology replicate in E coli
bull They have been engineered to optimize their use as vectors in DNA cloning
ndash To simplify working with plasmids their length is reduced
ndash Most plasmid vectors contain little more than the essential nucleotide
sequences required for their use in DNA cloning
bull Plasmid vectors are usually used as either cloning vectors expression vectorsssDNA vectors or transformation vectors
bull They are not used as library vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3953
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4053
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4253
Basic Characteristics of Cloning Vectors
Basic characteristics
1 Origin of replication (ori )
ndash An origin of replication is a sequence of DNA at which replication is initiated on achromosome plasmid or virus
bull small DNAs a single origin is sufficient ndash Larger DNAs have many origins and DNA replication is initiated at all of them
bull Determines the vector copy number
bull Determines the plasmids compatibility its ability to replicate in conjunction withanother plasmid within the same bacterial cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4353
Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4453
Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4553
Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4653
Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4753
Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4853
Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1053
Restriction Endonucleases
R-DNA technology uses type II ndash Why
1 Restriction activity and modifying activity are in separateenzymes
2 Restriction endonucleases cleave within the recognition site oradjacent to it
3 Do not need ATP for activity Needs only Mg++
4 Cuts in a precise manner very predictable
5 Short palindromic recognition sequence
Note
Over 1500 known over 150 available commercially
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1153
Restriction Endonucleases- Nomenclature
Named for bacterial genus species strain and type
xamp e coGenus E scherichia Species co li Strain ROrder discovered 1
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1253
Restriction Endonucleases- Nomenclature
eg Bam H1Bacillus amyloliquefaciens Strain H
First enzyme characterised
eg Hin dIIIHaemophilus influenzae Strain D - 3rd enzyme
eg Eco RI
Escherichia coli Strain R ndash 1st enzyme discovered
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1353
Restriction Endonucleases
Recognition sites have symmetry (palindromic)
ldquoAble was I ere I saw Elbardquo
Bam H1 site5rsquo-GGATCC-3rsquo
3rsquo-CCTAGG-5rsquo
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1453
Restriction Endonucleases
eg Bam H1
Cleavage site
GGATCCCCTAGG
Recognition sequence
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1553
Restriction Endonucleases
Cohesive end cutter or sticky end cutter
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1653
Restriction Endonucleases
eg Sma I = Serratia marcescens Enzyme 1
CCCGGG
Cleavage site
GGGCCC
Recognition sequence
BLUNT END CUTTEReg Sca1 EcoRV Nru1
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1753
Classification of Restriction Endonucleases
1 Sticky-end cutter vs blunt-end cutter
2 Frequent cutter vs rare cutter
Recognition sequence Cleavage frequency
4 bp (Alu1 HpaII HhaI) (144) = 1256
5 bp (Hga1) (145 ) = 110246 bp (BamH1 HinD111) (146 ) = 14096
7 bp (PpuM1) (147 ) = 116384
8 bp (Not1)
9 bp (AlwN1)10 bp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1853
983122983141983139983151983149983138983145983150983137983150983156 983140983150983137 983154983141983155983156983154983145983139983156983145983151983150
983141983150983140983151983150983157983139983148983141983137983155983141983155
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1953
Classification of Restriction Endonucleases
3 Ambiguous vs precise cutters
eg BsiE1
CGPuPyCGGCPyPuGC
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2053
Classification of Restriction Endonucleases
4 Isoshizomers
Some enzymes have the same target site (recognition site)
Two subtypes
Same target site ndash different cleavage site
eg Sma1 and Xma1
Same target site - same cleavage site
eg Aat1 and Stu1
BamH1 and Bst1
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2253
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2353
Restriction digestionCommercial restriction enzymes
1 Restriction enzymes are commercially available at 5 units per
microL concentration
2 I unit = 1 unit (U) is the amount if enzyme that catalyses the
conditions
3 Enzymes come in storage buffer containing EDTA DTT BSAand Glycerol
4 Too much enzyme in the reaction mix can cause star activity
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2453
Star activity
Loss of specificity of the restriction enzyme (typeII) underextreme non-standard conditions is called star activity
Under such conditions the restriction enzyme may cleave similarbut non identical target sequences
Non-standard conditions include1 high glycerol concentration gt5 vv
2 gt100 units per ug of DNA
3 Low ionic strength buffer lt25mM (usually is 100-150 mM)
4 high pH (gt 80) usually pH is around 75 presence of organic solvents DMSO ethanol PEGdimethyl acetamide
6 Cations other than Mg++ eg Cu++ Co++Zn++ Mn++
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2553
Restriction digestion
Stopping the Restriction digestion
1 Heat inactivation of restriction enzymes
Place reaction mix in a waterbath set at 65oCNote not all Restriction enzymes can be heat inactivated egPvu1 Bgl II BamH1 Acc1
2 Add 2ul of 25 mM EDTAEDTA chelates the Mg++ and makes it unavailable to theRestriction Endonuclease
3 Phenol-chloroform extraction purificationRemoves all proteins including the enzymes
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2653
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Single Digests
EcoRI BamHI NcoI
Number offragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2753
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
U n d i g e s t e d
N c o I
EcoRI BamHI NcoI
Numberof
fragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2853
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Double Digests
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2953
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
E c o R I
N c o I
U n d i g e s t e d
B a m H I
N c o I
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3053
SummaryRestriction Digestion is the process of cutting DNA molecules into smaller pieces
with special enzymes called Restriction Endonucleases (sometimes just called
Restriction Enzymes or REs) These special enzymes recognize specificsequences in the DNA molecule (for example GATATC) wherever that sequenceoccurs in the DNA
httphigheredmcgraw-hillcomsites9834092339student_view0chapter18restriction_endonucleaseshtml
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3153
Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3253
So what are vectors
bull Vector (molecular biology) vehicle used to transfer genetic material to a target
cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3353
So what are vectors
ndash Plasmid vector
ndash Binary vector a cloning vector used to generate transgenic plants
ndash Cloning vector
ndash Expression vector a plasmid specifically used for protein expression in thetarget
ndash Shuttle vector a vector (usually a plasmid) constructed so that it canpropagate in two different host species
ndash Viral vector a virus modified to deliver foreign genetic material into a cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3453
Vectors can be classified based on
bull The purpose of the vector ndash eg cloning library construction ssDNA production mRNA synthesis expression
multipurpose etc
bull Origin of vector ndash Plasmid ndash Bacteriophage ndash Cosmid ndash Phagemid ndash YAC BAC
bull The host organism that the vector is propagated in ndash Bacteria (bacterial origin of replication) ndash Yeast (yeast origin of replication) ndash Shuttle vector (both origins)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3553
Types of vectors
Vector Target fragment length
Plasmid 0-10 kb (total size up to 15 kb)
Cosmid 10-40 kb
P1 artificial chromosome (PAC) 130-150 kbBacterial artificial chromosome (BAC) About 300 kb
Yeast artificial chromosome (YAC) 200 kb to 2 Mb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3653
What vectors will I be covering
Vectors
bull Plasmid vectorso Cloning vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3753
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3853
Plasmid Vectors
bull Plasmids most commonly used in recombinant DNA technology replicate in E coli
bull They have been engineered to optimize their use as vectors in DNA cloning
ndash To simplify working with plasmids their length is reduced
ndash Most plasmid vectors contain little more than the essential nucleotide
sequences required for their use in DNA cloning
bull Plasmid vectors are usually used as either cloning vectors expression vectorsssDNA vectors or transformation vectors
bull They are not used as library vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3953
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4053
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4253
Basic Characteristics of Cloning Vectors
Basic characteristics
1 Origin of replication (ori )
ndash An origin of replication is a sequence of DNA at which replication is initiated on achromosome plasmid or virus
bull small DNAs a single origin is sufficient ndash Larger DNAs have many origins and DNA replication is initiated at all of them
bull Determines the vector copy number
bull Determines the plasmids compatibility its ability to replicate in conjunction withanother plasmid within the same bacterial cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4353
Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4453
Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4553
Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4653
Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4753
Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4853
Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1153
Restriction Endonucleases- Nomenclature
Named for bacterial genus species strain and type
xamp e coGenus E scherichia Species co li Strain ROrder discovered 1
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1253
Restriction Endonucleases- Nomenclature
eg Bam H1Bacillus amyloliquefaciens Strain H
First enzyme characterised
eg Hin dIIIHaemophilus influenzae Strain D - 3rd enzyme
eg Eco RI
Escherichia coli Strain R ndash 1st enzyme discovered
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1353
Restriction Endonucleases
Recognition sites have symmetry (palindromic)
ldquoAble was I ere I saw Elbardquo
Bam H1 site5rsquo-GGATCC-3rsquo
3rsquo-CCTAGG-5rsquo
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1453
Restriction Endonucleases
eg Bam H1
Cleavage site
GGATCCCCTAGG
Recognition sequence
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1553
Restriction Endonucleases
Cohesive end cutter or sticky end cutter
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1653
Restriction Endonucleases
eg Sma I = Serratia marcescens Enzyme 1
CCCGGG
Cleavage site
GGGCCC
Recognition sequence
BLUNT END CUTTEReg Sca1 EcoRV Nru1
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1753
Classification of Restriction Endonucleases
1 Sticky-end cutter vs blunt-end cutter
2 Frequent cutter vs rare cutter
Recognition sequence Cleavage frequency
4 bp (Alu1 HpaII HhaI) (144) = 1256
5 bp (Hga1) (145 ) = 110246 bp (BamH1 HinD111) (146 ) = 14096
7 bp (PpuM1) (147 ) = 116384
8 bp (Not1)
9 bp (AlwN1)10 bp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1853
983122983141983139983151983149983138983145983150983137983150983156 983140983150983137 983154983141983155983156983154983145983139983156983145983151983150
983141983150983140983151983150983157983139983148983141983137983155983141983155
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1953
Classification of Restriction Endonucleases
3 Ambiguous vs precise cutters
eg BsiE1
CGPuPyCGGCPyPuGC
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2053
Classification of Restriction Endonucleases
4 Isoshizomers
Some enzymes have the same target site (recognition site)
Two subtypes
Same target site ndash different cleavage site
eg Sma1 and Xma1
Same target site - same cleavage site
eg Aat1 and Stu1
BamH1 and Bst1
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2253
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2353
Restriction digestionCommercial restriction enzymes
1 Restriction enzymes are commercially available at 5 units per
microL concentration
2 I unit = 1 unit (U) is the amount if enzyme that catalyses the
conditions
3 Enzymes come in storage buffer containing EDTA DTT BSAand Glycerol
4 Too much enzyme in the reaction mix can cause star activity
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2453
Star activity
Loss of specificity of the restriction enzyme (typeII) underextreme non-standard conditions is called star activity
Under such conditions the restriction enzyme may cleave similarbut non identical target sequences
Non-standard conditions include1 high glycerol concentration gt5 vv
2 gt100 units per ug of DNA
3 Low ionic strength buffer lt25mM (usually is 100-150 mM)
4 high pH (gt 80) usually pH is around 75 presence of organic solvents DMSO ethanol PEGdimethyl acetamide
6 Cations other than Mg++ eg Cu++ Co++Zn++ Mn++
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2553
Restriction digestion
Stopping the Restriction digestion
1 Heat inactivation of restriction enzymes
Place reaction mix in a waterbath set at 65oCNote not all Restriction enzymes can be heat inactivated egPvu1 Bgl II BamH1 Acc1
2 Add 2ul of 25 mM EDTAEDTA chelates the Mg++ and makes it unavailable to theRestriction Endonuclease
3 Phenol-chloroform extraction purificationRemoves all proteins including the enzymes
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2653
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Single Digests
EcoRI BamHI NcoI
Number offragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2753
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
U n d i g e s t e d
N c o I
EcoRI BamHI NcoI
Numberof
fragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2853
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Double Digests
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2953
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
E c o R I
N c o I
U n d i g e s t e d
B a m H I
N c o I
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3053
SummaryRestriction Digestion is the process of cutting DNA molecules into smaller pieces
with special enzymes called Restriction Endonucleases (sometimes just called
Restriction Enzymes or REs) These special enzymes recognize specificsequences in the DNA molecule (for example GATATC) wherever that sequenceoccurs in the DNA
httphigheredmcgraw-hillcomsites9834092339student_view0chapter18restriction_endonucleaseshtml
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3153
Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3253
So what are vectors
bull Vector (molecular biology) vehicle used to transfer genetic material to a target
cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3353
So what are vectors
ndash Plasmid vector
ndash Binary vector a cloning vector used to generate transgenic plants
ndash Cloning vector
ndash Expression vector a plasmid specifically used for protein expression in thetarget
ndash Shuttle vector a vector (usually a plasmid) constructed so that it canpropagate in two different host species
ndash Viral vector a virus modified to deliver foreign genetic material into a cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3453
Vectors can be classified based on
bull The purpose of the vector ndash eg cloning library construction ssDNA production mRNA synthesis expression
multipurpose etc
bull Origin of vector ndash Plasmid ndash Bacteriophage ndash Cosmid ndash Phagemid ndash YAC BAC
bull The host organism that the vector is propagated in ndash Bacteria (bacterial origin of replication) ndash Yeast (yeast origin of replication) ndash Shuttle vector (both origins)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3553
Types of vectors
Vector Target fragment length
Plasmid 0-10 kb (total size up to 15 kb)
Cosmid 10-40 kb
P1 artificial chromosome (PAC) 130-150 kbBacterial artificial chromosome (BAC) About 300 kb
Yeast artificial chromosome (YAC) 200 kb to 2 Mb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3653
What vectors will I be covering
Vectors
bull Plasmid vectorso Cloning vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3753
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3853
Plasmid Vectors
bull Plasmids most commonly used in recombinant DNA technology replicate in E coli
bull They have been engineered to optimize their use as vectors in DNA cloning
ndash To simplify working with plasmids their length is reduced
ndash Most plasmid vectors contain little more than the essential nucleotide
sequences required for their use in DNA cloning
bull Plasmid vectors are usually used as either cloning vectors expression vectorsssDNA vectors or transformation vectors
bull They are not used as library vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3953
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4053
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4253
Basic Characteristics of Cloning Vectors
Basic characteristics
1 Origin of replication (ori )
ndash An origin of replication is a sequence of DNA at which replication is initiated on achromosome plasmid or virus
bull small DNAs a single origin is sufficient ndash Larger DNAs have many origins and DNA replication is initiated at all of them
bull Determines the vector copy number
bull Determines the plasmids compatibility its ability to replicate in conjunction withanother plasmid within the same bacterial cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4353
Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4453
Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4553
Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4653
Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4753
Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4853
Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1253
Restriction Endonucleases- Nomenclature
eg Bam H1Bacillus amyloliquefaciens Strain H
First enzyme characterised
eg Hin dIIIHaemophilus influenzae Strain D - 3rd enzyme
eg Eco RI
Escherichia coli Strain R ndash 1st enzyme discovered
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1353
Restriction Endonucleases
Recognition sites have symmetry (palindromic)
ldquoAble was I ere I saw Elbardquo
Bam H1 site5rsquo-GGATCC-3rsquo
3rsquo-CCTAGG-5rsquo
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1453
Restriction Endonucleases
eg Bam H1
Cleavage site
GGATCCCCTAGG
Recognition sequence
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1553
Restriction Endonucleases
Cohesive end cutter or sticky end cutter
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1653
Restriction Endonucleases
eg Sma I = Serratia marcescens Enzyme 1
CCCGGG
Cleavage site
GGGCCC
Recognition sequence
BLUNT END CUTTEReg Sca1 EcoRV Nru1
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1753
Classification of Restriction Endonucleases
1 Sticky-end cutter vs blunt-end cutter
2 Frequent cutter vs rare cutter
Recognition sequence Cleavage frequency
4 bp (Alu1 HpaII HhaI) (144) = 1256
5 bp (Hga1) (145 ) = 110246 bp (BamH1 HinD111) (146 ) = 14096
7 bp (PpuM1) (147 ) = 116384
8 bp (Not1)
9 bp (AlwN1)10 bp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1853
983122983141983139983151983149983138983145983150983137983150983156 983140983150983137 983154983141983155983156983154983145983139983156983145983151983150
983141983150983140983151983150983157983139983148983141983137983155983141983155
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1953
Classification of Restriction Endonucleases
3 Ambiguous vs precise cutters
eg BsiE1
CGPuPyCGGCPyPuGC
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2053
Classification of Restriction Endonucleases
4 Isoshizomers
Some enzymes have the same target site (recognition site)
Two subtypes
Same target site ndash different cleavage site
eg Sma1 and Xma1
Same target site - same cleavage site
eg Aat1 and Stu1
BamH1 and Bst1
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2253
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2353
Restriction digestionCommercial restriction enzymes
1 Restriction enzymes are commercially available at 5 units per
microL concentration
2 I unit = 1 unit (U) is the amount if enzyme that catalyses the
conditions
3 Enzymes come in storage buffer containing EDTA DTT BSAand Glycerol
4 Too much enzyme in the reaction mix can cause star activity
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2453
Star activity
Loss of specificity of the restriction enzyme (typeII) underextreme non-standard conditions is called star activity
Under such conditions the restriction enzyme may cleave similarbut non identical target sequences
Non-standard conditions include1 high glycerol concentration gt5 vv
2 gt100 units per ug of DNA
3 Low ionic strength buffer lt25mM (usually is 100-150 mM)
4 high pH (gt 80) usually pH is around 75 presence of organic solvents DMSO ethanol PEGdimethyl acetamide
6 Cations other than Mg++ eg Cu++ Co++Zn++ Mn++
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2553
Restriction digestion
Stopping the Restriction digestion
1 Heat inactivation of restriction enzymes
Place reaction mix in a waterbath set at 65oCNote not all Restriction enzymes can be heat inactivated egPvu1 Bgl II BamH1 Acc1
2 Add 2ul of 25 mM EDTAEDTA chelates the Mg++ and makes it unavailable to theRestriction Endonuclease
3 Phenol-chloroform extraction purificationRemoves all proteins including the enzymes
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2653
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Single Digests
EcoRI BamHI NcoI
Number offragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2753
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
U n d i g e s t e d
N c o I
EcoRI BamHI NcoI
Numberof
fragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2853
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Double Digests
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2953
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
E c o R I
N c o I
U n d i g e s t e d
B a m H I
N c o I
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3053
SummaryRestriction Digestion is the process of cutting DNA molecules into smaller pieces
with special enzymes called Restriction Endonucleases (sometimes just called
Restriction Enzymes or REs) These special enzymes recognize specificsequences in the DNA molecule (for example GATATC) wherever that sequenceoccurs in the DNA
httphigheredmcgraw-hillcomsites9834092339student_view0chapter18restriction_endonucleaseshtml
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3153
Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3253
So what are vectors
bull Vector (molecular biology) vehicle used to transfer genetic material to a target
cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3353
So what are vectors
ndash Plasmid vector
ndash Binary vector a cloning vector used to generate transgenic plants
ndash Cloning vector
ndash Expression vector a plasmid specifically used for protein expression in thetarget
ndash Shuttle vector a vector (usually a plasmid) constructed so that it canpropagate in two different host species
ndash Viral vector a virus modified to deliver foreign genetic material into a cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3453
Vectors can be classified based on
bull The purpose of the vector ndash eg cloning library construction ssDNA production mRNA synthesis expression
multipurpose etc
bull Origin of vector ndash Plasmid ndash Bacteriophage ndash Cosmid ndash Phagemid ndash YAC BAC
bull The host organism that the vector is propagated in ndash Bacteria (bacterial origin of replication) ndash Yeast (yeast origin of replication) ndash Shuttle vector (both origins)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3553
Types of vectors
Vector Target fragment length
Plasmid 0-10 kb (total size up to 15 kb)
Cosmid 10-40 kb
P1 artificial chromosome (PAC) 130-150 kbBacterial artificial chromosome (BAC) About 300 kb
Yeast artificial chromosome (YAC) 200 kb to 2 Mb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3653
What vectors will I be covering
Vectors
bull Plasmid vectorso Cloning vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3753
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3853
Plasmid Vectors
bull Plasmids most commonly used in recombinant DNA technology replicate in E coli
bull They have been engineered to optimize their use as vectors in DNA cloning
ndash To simplify working with plasmids their length is reduced
ndash Most plasmid vectors contain little more than the essential nucleotide
sequences required for their use in DNA cloning
bull Plasmid vectors are usually used as either cloning vectors expression vectorsssDNA vectors or transformation vectors
bull They are not used as library vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3953
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4053
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4253
Basic Characteristics of Cloning Vectors
Basic characteristics
1 Origin of replication (ori )
ndash An origin of replication is a sequence of DNA at which replication is initiated on achromosome plasmid or virus
bull small DNAs a single origin is sufficient ndash Larger DNAs have many origins and DNA replication is initiated at all of them
bull Determines the vector copy number
bull Determines the plasmids compatibility its ability to replicate in conjunction withanother plasmid within the same bacterial cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4353
Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4453
Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4553
Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4653
Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4753
Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4853
Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1353
Restriction Endonucleases
Recognition sites have symmetry (palindromic)
ldquoAble was I ere I saw Elbardquo
Bam H1 site5rsquo-GGATCC-3rsquo
3rsquo-CCTAGG-5rsquo
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1453
Restriction Endonucleases
eg Bam H1
Cleavage site
GGATCCCCTAGG
Recognition sequence
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1553
Restriction Endonucleases
Cohesive end cutter or sticky end cutter
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1653
Restriction Endonucleases
eg Sma I = Serratia marcescens Enzyme 1
CCCGGG
Cleavage site
GGGCCC
Recognition sequence
BLUNT END CUTTEReg Sca1 EcoRV Nru1
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1753
Classification of Restriction Endonucleases
1 Sticky-end cutter vs blunt-end cutter
2 Frequent cutter vs rare cutter
Recognition sequence Cleavage frequency
4 bp (Alu1 HpaII HhaI) (144) = 1256
5 bp (Hga1) (145 ) = 110246 bp (BamH1 HinD111) (146 ) = 14096
7 bp (PpuM1) (147 ) = 116384
8 bp (Not1)
9 bp (AlwN1)10 bp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1853
983122983141983139983151983149983138983145983150983137983150983156 983140983150983137 983154983141983155983156983154983145983139983156983145983151983150
983141983150983140983151983150983157983139983148983141983137983155983141983155
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1953
Classification of Restriction Endonucleases
3 Ambiguous vs precise cutters
eg BsiE1
CGPuPyCGGCPyPuGC
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2053
Classification of Restriction Endonucleases
4 Isoshizomers
Some enzymes have the same target site (recognition site)
Two subtypes
Same target site ndash different cleavage site
eg Sma1 and Xma1
Same target site - same cleavage site
eg Aat1 and Stu1
BamH1 and Bst1
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2253
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2353
Restriction digestionCommercial restriction enzymes
1 Restriction enzymes are commercially available at 5 units per
microL concentration
2 I unit = 1 unit (U) is the amount if enzyme that catalyses the
conditions
3 Enzymes come in storage buffer containing EDTA DTT BSAand Glycerol
4 Too much enzyme in the reaction mix can cause star activity
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2453
Star activity
Loss of specificity of the restriction enzyme (typeII) underextreme non-standard conditions is called star activity
Under such conditions the restriction enzyme may cleave similarbut non identical target sequences
Non-standard conditions include1 high glycerol concentration gt5 vv
2 gt100 units per ug of DNA
3 Low ionic strength buffer lt25mM (usually is 100-150 mM)
4 high pH (gt 80) usually pH is around 75 presence of organic solvents DMSO ethanol PEGdimethyl acetamide
6 Cations other than Mg++ eg Cu++ Co++Zn++ Mn++
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2553
Restriction digestion
Stopping the Restriction digestion
1 Heat inactivation of restriction enzymes
Place reaction mix in a waterbath set at 65oCNote not all Restriction enzymes can be heat inactivated egPvu1 Bgl II BamH1 Acc1
2 Add 2ul of 25 mM EDTAEDTA chelates the Mg++ and makes it unavailable to theRestriction Endonuclease
3 Phenol-chloroform extraction purificationRemoves all proteins including the enzymes
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2653
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Single Digests
EcoRI BamHI NcoI
Number offragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2753
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
U n d i g e s t e d
N c o I
EcoRI BamHI NcoI
Numberof
fragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2853
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Double Digests
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2953
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
E c o R I
N c o I
U n d i g e s t e d
B a m H I
N c o I
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3053
SummaryRestriction Digestion is the process of cutting DNA molecules into smaller pieces
with special enzymes called Restriction Endonucleases (sometimes just called
Restriction Enzymes or REs) These special enzymes recognize specificsequences in the DNA molecule (for example GATATC) wherever that sequenceoccurs in the DNA
httphigheredmcgraw-hillcomsites9834092339student_view0chapter18restriction_endonucleaseshtml
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3153
Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3253
So what are vectors
bull Vector (molecular biology) vehicle used to transfer genetic material to a target
cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3353
So what are vectors
ndash Plasmid vector
ndash Binary vector a cloning vector used to generate transgenic plants
ndash Cloning vector
ndash Expression vector a plasmid specifically used for protein expression in thetarget
ndash Shuttle vector a vector (usually a plasmid) constructed so that it canpropagate in two different host species
ndash Viral vector a virus modified to deliver foreign genetic material into a cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3453
Vectors can be classified based on
bull The purpose of the vector ndash eg cloning library construction ssDNA production mRNA synthesis expression
multipurpose etc
bull Origin of vector ndash Plasmid ndash Bacteriophage ndash Cosmid ndash Phagemid ndash YAC BAC
bull The host organism that the vector is propagated in ndash Bacteria (bacterial origin of replication) ndash Yeast (yeast origin of replication) ndash Shuttle vector (both origins)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3553
Types of vectors
Vector Target fragment length
Plasmid 0-10 kb (total size up to 15 kb)
Cosmid 10-40 kb
P1 artificial chromosome (PAC) 130-150 kbBacterial artificial chromosome (BAC) About 300 kb
Yeast artificial chromosome (YAC) 200 kb to 2 Mb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3653
What vectors will I be covering
Vectors
bull Plasmid vectorso Cloning vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3753
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3853
Plasmid Vectors
bull Plasmids most commonly used in recombinant DNA technology replicate in E coli
bull They have been engineered to optimize their use as vectors in DNA cloning
ndash To simplify working with plasmids their length is reduced
ndash Most plasmid vectors contain little more than the essential nucleotide
sequences required for their use in DNA cloning
bull Plasmid vectors are usually used as either cloning vectors expression vectorsssDNA vectors or transformation vectors
bull They are not used as library vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3953
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4053
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4253
Basic Characteristics of Cloning Vectors
Basic characteristics
1 Origin of replication (ori )
ndash An origin of replication is a sequence of DNA at which replication is initiated on achromosome plasmid or virus
bull small DNAs a single origin is sufficient ndash Larger DNAs have many origins and DNA replication is initiated at all of them
bull Determines the vector copy number
bull Determines the plasmids compatibility its ability to replicate in conjunction withanother plasmid within the same bacterial cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4353
Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4453
Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4553
Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4653
Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4753
Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4853
Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1453
Restriction Endonucleases
eg Bam H1
Cleavage site
GGATCCCCTAGG
Recognition sequence
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1553
Restriction Endonucleases
Cohesive end cutter or sticky end cutter
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1653
Restriction Endonucleases
eg Sma I = Serratia marcescens Enzyme 1
CCCGGG
Cleavage site
GGGCCC
Recognition sequence
BLUNT END CUTTEReg Sca1 EcoRV Nru1
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1753
Classification of Restriction Endonucleases
1 Sticky-end cutter vs blunt-end cutter
2 Frequent cutter vs rare cutter
Recognition sequence Cleavage frequency
4 bp (Alu1 HpaII HhaI) (144) = 1256
5 bp (Hga1) (145 ) = 110246 bp (BamH1 HinD111) (146 ) = 14096
7 bp (PpuM1) (147 ) = 116384
8 bp (Not1)
9 bp (AlwN1)10 bp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1853
983122983141983139983151983149983138983145983150983137983150983156 983140983150983137 983154983141983155983156983154983145983139983156983145983151983150
983141983150983140983151983150983157983139983148983141983137983155983141983155
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1953
Classification of Restriction Endonucleases
3 Ambiguous vs precise cutters
eg BsiE1
CGPuPyCGGCPyPuGC
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2053
Classification of Restriction Endonucleases
4 Isoshizomers
Some enzymes have the same target site (recognition site)
Two subtypes
Same target site ndash different cleavage site
eg Sma1 and Xma1
Same target site - same cleavage site
eg Aat1 and Stu1
BamH1 and Bst1
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2253
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2353
Restriction digestionCommercial restriction enzymes
1 Restriction enzymes are commercially available at 5 units per
microL concentration
2 I unit = 1 unit (U) is the amount if enzyme that catalyses the
conditions
3 Enzymes come in storage buffer containing EDTA DTT BSAand Glycerol
4 Too much enzyme in the reaction mix can cause star activity
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2453
Star activity
Loss of specificity of the restriction enzyme (typeII) underextreme non-standard conditions is called star activity
Under such conditions the restriction enzyme may cleave similarbut non identical target sequences
Non-standard conditions include1 high glycerol concentration gt5 vv
2 gt100 units per ug of DNA
3 Low ionic strength buffer lt25mM (usually is 100-150 mM)
4 high pH (gt 80) usually pH is around 75 presence of organic solvents DMSO ethanol PEGdimethyl acetamide
6 Cations other than Mg++ eg Cu++ Co++Zn++ Mn++
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2553
Restriction digestion
Stopping the Restriction digestion
1 Heat inactivation of restriction enzymes
Place reaction mix in a waterbath set at 65oCNote not all Restriction enzymes can be heat inactivated egPvu1 Bgl II BamH1 Acc1
2 Add 2ul of 25 mM EDTAEDTA chelates the Mg++ and makes it unavailable to theRestriction Endonuclease
3 Phenol-chloroform extraction purificationRemoves all proteins including the enzymes
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2653
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Single Digests
EcoRI BamHI NcoI
Number offragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2753
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
U n d i g e s t e d
N c o I
EcoRI BamHI NcoI
Numberof
fragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2853
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Double Digests
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2953
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
E c o R I
N c o I
U n d i g e s t e d
B a m H I
N c o I
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3053
SummaryRestriction Digestion is the process of cutting DNA molecules into smaller pieces
with special enzymes called Restriction Endonucleases (sometimes just called
Restriction Enzymes or REs) These special enzymes recognize specificsequences in the DNA molecule (for example GATATC) wherever that sequenceoccurs in the DNA
httphigheredmcgraw-hillcomsites9834092339student_view0chapter18restriction_endonucleaseshtml
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3153
Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3253
So what are vectors
bull Vector (molecular biology) vehicle used to transfer genetic material to a target
cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3353
So what are vectors
ndash Plasmid vector
ndash Binary vector a cloning vector used to generate transgenic plants
ndash Cloning vector
ndash Expression vector a plasmid specifically used for protein expression in thetarget
ndash Shuttle vector a vector (usually a plasmid) constructed so that it canpropagate in two different host species
ndash Viral vector a virus modified to deliver foreign genetic material into a cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3453
Vectors can be classified based on
bull The purpose of the vector ndash eg cloning library construction ssDNA production mRNA synthesis expression
multipurpose etc
bull Origin of vector ndash Plasmid ndash Bacteriophage ndash Cosmid ndash Phagemid ndash YAC BAC
bull The host organism that the vector is propagated in ndash Bacteria (bacterial origin of replication) ndash Yeast (yeast origin of replication) ndash Shuttle vector (both origins)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3553
Types of vectors
Vector Target fragment length
Plasmid 0-10 kb (total size up to 15 kb)
Cosmid 10-40 kb
P1 artificial chromosome (PAC) 130-150 kbBacterial artificial chromosome (BAC) About 300 kb
Yeast artificial chromosome (YAC) 200 kb to 2 Mb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3653
What vectors will I be covering
Vectors
bull Plasmid vectorso Cloning vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3753
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3853
Plasmid Vectors
bull Plasmids most commonly used in recombinant DNA technology replicate in E coli
bull They have been engineered to optimize their use as vectors in DNA cloning
ndash To simplify working with plasmids their length is reduced
ndash Most plasmid vectors contain little more than the essential nucleotide
sequences required for their use in DNA cloning
bull Plasmid vectors are usually used as either cloning vectors expression vectorsssDNA vectors or transformation vectors
bull They are not used as library vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3953
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4053
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4253
Basic Characteristics of Cloning Vectors
Basic characteristics
1 Origin of replication (ori )
ndash An origin of replication is a sequence of DNA at which replication is initiated on achromosome plasmid or virus
bull small DNAs a single origin is sufficient ndash Larger DNAs have many origins and DNA replication is initiated at all of them
bull Determines the vector copy number
bull Determines the plasmids compatibility its ability to replicate in conjunction withanother plasmid within the same bacterial cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4353
Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4453
Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4553
Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4653
Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4753
Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4853
Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1553
Restriction Endonucleases
Cohesive end cutter or sticky end cutter
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1653
Restriction Endonucleases
eg Sma I = Serratia marcescens Enzyme 1
CCCGGG
Cleavage site
GGGCCC
Recognition sequence
BLUNT END CUTTEReg Sca1 EcoRV Nru1
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1753
Classification of Restriction Endonucleases
1 Sticky-end cutter vs blunt-end cutter
2 Frequent cutter vs rare cutter
Recognition sequence Cleavage frequency
4 bp (Alu1 HpaII HhaI) (144) = 1256
5 bp (Hga1) (145 ) = 110246 bp (BamH1 HinD111) (146 ) = 14096
7 bp (PpuM1) (147 ) = 116384
8 bp (Not1)
9 bp (AlwN1)10 bp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1853
983122983141983139983151983149983138983145983150983137983150983156 983140983150983137 983154983141983155983156983154983145983139983156983145983151983150
983141983150983140983151983150983157983139983148983141983137983155983141983155
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1953
Classification of Restriction Endonucleases
3 Ambiguous vs precise cutters
eg BsiE1
CGPuPyCGGCPyPuGC
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2053
Classification of Restriction Endonucleases
4 Isoshizomers
Some enzymes have the same target site (recognition site)
Two subtypes
Same target site ndash different cleavage site
eg Sma1 and Xma1
Same target site - same cleavage site
eg Aat1 and Stu1
BamH1 and Bst1
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2253
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2353
Restriction digestionCommercial restriction enzymes
1 Restriction enzymes are commercially available at 5 units per
microL concentration
2 I unit = 1 unit (U) is the amount if enzyme that catalyses the
conditions
3 Enzymes come in storage buffer containing EDTA DTT BSAand Glycerol
4 Too much enzyme in the reaction mix can cause star activity
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2453
Star activity
Loss of specificity of the restriction enzyme (typeII) underextreme non-standard conditions is called star activity
Under such conditions the restriction enzyme may cleave similarbut non identical target sequences
Non-standard conditions include1 high glycerol concentration gt5 vv
2 gt100 units per ug of DNA
3 Low ionic strength buffer lt25mM (usually is 100-150 mM)
4 high pH (gt 80) usually pH is around 75 presence of organic solvents DMSO ethanol PEGdimethyl acetamide
6 Cations other than Mg++ eg Cu++ Co++Zn++ Mn++
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2553
Restriction digestion
Stopping the Restriction digestion
1 Heat inactivation of restriction enzymes
Place reaction mix in a waterbath set at 65oCNote not all Restriction enzymes can be heat inactivated egPvu1 Bgl II BamH1 Acc1
2 Add 2ul of 25 mM EDTAEDTA chelates the Mg++ and makes it unavailable to theRestriction Endonuclease
3 Phenol-chloroform extraction purificationRemoves all proteins including the enzymes
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2653
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Single Digests
EcoRI BamHI NcoI
Number offragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2753
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
U n d i g e s t e d
N c o I
EcoRI BamHI NcoI
Numberof
fragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2853
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Double Digests
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2953
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
E c o R I
N c o I
U n d i g e s t e d
B a m H I
N c o I
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3053
SummaryRestriction Digestion is the process of cutting DNA molecules into smaller pieces
with special enzymes called Restriction Endonucleases (sometimes just called
Restriction Enzymes or REs) These special enzymes recognize specificsequences in the DNA molecule (for example GATATC) wherever that sequenceoccurs in the DNA
httphigheredmcgraw-hillcomsites9834092339student_view0chapter18restriction_endonucleaseshtml
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3153
Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3253
So what are vectors
bull Vector (molecular biology) vehicle used to transfer genetic material to a target
cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3353
So what are vectors
ndash Plasmid vector
ndash Binary vector a cloning vector used to generate transgenic plants
ndash Cloning vector
ndash Expression vector a plasmid specifically used for protein expression in thetarget
ndash Shuttle vector a vector (usually a plasmid) constructed so that it canpropagate in two different host species
ndash Viral vector a virus modified to deliver foreign genetic material into a cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3453
Vectors can be classified based on
bull The purpose of the vector ndash eg cloning library construction ssDNA production mRNA synthesis expression
multipurpose etc
bull Origin of vector ndash Plasmid ndash Bacteriophage ndash Cosmid ndash Phagemid ndash YAC BAC
bull The host organism that the vector is propagated in ndash Bacteria (bacterial origin of replication) ndash Yeast (yeast origin of replication) ndash Shuttle vector (both origins)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3553
Types of vectors
Vector Target fragment length
Plasmid 0-10 kb (total size up to 15 kb)
Cosmid 10-40 kb
P1 artificial chromosome (PAC) 130-150 kbBacterial artificial chromosome (BAC) About 300 kb
Yeast artificial chromosome (YAC) 200 kb to 2 Mb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3653
What vectors will I be covering
Vectors
bull Plasmid vectorso Cloning vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3753
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3853
Plasmid Vectors
bull Plasmids most commonly used in recombinant DNA technology replicate in E coli
bull They have been engineered to optimize their use as vectors in DNA cloning
ndash To simplify working with plasmids their length is reduced
ndash Most plasmid vectors contain little more than the essential nucleotide
sequences required for their use in DNA cloning
bull Plasmid vectors are usually used as either cloning vectors expression vectorsssDNA vectors or transformation vectors
bull They are not used as library vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3953
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4053
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4253
Basic Characteristics of Cloning Vectors
Basic characteristics
1 Origin of replication (ori )
ndash An origin of replication is a sequence of DNA at which replication is initiated on achromosome plasmid or virus
bull small DNAs a single origin is sufficient ndash Larger DNAs have many origins and DNA replication is initiated at all of them
bull Determines the vector copy number
bull Determines the plasmids compatibility its ability to replicate in conjunction withanother plasmid within the same bacterial cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4353
Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4453
Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4553
Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4653
Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4753
Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4853
Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1653
Restriction Endonucleases
eg Sma I = Serratia marcescens Enzyme 1
CCCGGG
Cleavage site
GGGCCC
Recognition sequence
BLUNT END CUTTEReg Sca1 EcoRV Nru1
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1753
Classification of Restriction Endonucleases
1 Sticky-end cutter vs blunt-end cutter
2 Frequent cutter vs rare cutter
Recognition sequence Cleavage frequency
4 bp (Alu1 HpaII HhaI) (144) = 1256
5 bp (Hga1) (145 ) = 110246 bp (BamH1 HinD111) (146 ) = 14096
7 bp (PpuM1) (147 ) = 116384
8 bp (Not1)
9 bp (AlwN1)10 bp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1853
983122983141983139983151983149983138983145983150983137983150983156 983140983150983137 983154983141983155983156983154983145983139983156983145983151983150
983141983150983140983151983150983157983139983148983141983137983155983141983155
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1953
Classification of Restriction Endonucleases
3 Ambiguous vs precise cutters
eg BsiE1
CGPuPyCGGCPyPuGC
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2053
Classification of Restriction Endonucleases
4 Isoshizomers
Some enzymes have the same target site (recognition site)
Two subtypes
Same target site ndash different cleavage site
eg Sma1 and Xma1
Same target site - same cleavage site
eg Aat1 and Stu1
BamH1 and Bst1
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2253
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2353
Restriction digestionCommercial restriction enzymes
1 Restriction enzymes are commercially available at 5 units per
microL concentration
2 I unit = 1 unit (U) is the amount if enzyme that catalyses the
conditions
3 Enzymes come in storage buffer containing EDTA DTT BSAand Glycerol
4 Too much enzyme in the reaction mix can cause star activity
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2453
Star activity
Loss of specificity of the restriction enzyme (typeII) underextreme non-standard conditions is called star activity
Under such conditions the restriction enzyme may cleave similarbut non identical target sequences
Non-standard conditions include1 high glycerol concentration gt5 vv
2 gt100 units per ug of DNA
3 Low ionic strength buffer lt25mM (usually is 100-150 mM)
4 high pH (gt 80) usually pH is around 75 presence of organic solvents DMSO ethanol PEGdimethyl acetamide
6 Cations other than Mg++ eg Cu++ Co++Zn++ Mn++
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2553
Restriction digestion
Stopping the Restriction digestion
1 Heat inactivation of restriction enzymes
Place reaction mix in a waterbath set at 65oCNote not all Restriction enzymes can be heat inactivated egPvu1 Bgl II BamH1 Acc1
2 Add 2ul of 25 mM EDTAEDTA chelates the Mg++ and makes it unavailable to theRestriction Endonuclease
3 Phenol-chloroform extraction purificationRemoves all proteins including the enzymes
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2653
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Single Digests
EcoRI BamHI NcoI
Number offragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2753
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
U n d i g e s t e d
N c o I
EcoRI BamHI NcoI
Numberof
fragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2853
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Double Digests
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2953
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
E c o R I
N c o I
U n d i g e s t e d
B a m H I
N c o I
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3053
SummaryRestriction Digestion is the process of cutting DNA molecules into smaller pieces
with special enzymes called Restriction Endonucleases (sometimes just called
Restriction Enzymes or REs) These special enzymes recognize specificsequences in the DNA molecule (for example GATATC) wherever that sequenceoccurs in the DNA
httphigheredmcgraw-hillcomsites9834092339student_view0chapter18restriction_endonucleaseshtml
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3153
Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3253
So what are vectors
bull Vector (molecular biology) vehicle used to transfer genetic material to a target
cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3353
So what are vectors
ndash Plasmid vector
ndash Binary vector a cloning vector used to generate transgenic plants
ndash Cloning vector
ndash Expression vector a plasmid specifically used for protein expression in thetarget
ndash Shuttle vector a vector (usually a plasmid) constructed so that it canpropagate in two different host species
ndash Viral vector a virus modified to deliver foreign genetic material into a cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3453
Vectors can be classified based on
bull The purpose of the vector ndash eg cloning library construction ssDNA production mRNA synthesis expression
multipurpose etc
bull Origin of vector ndash Plasmid ndash Bacteriophage ndash Cosmid ndash Phagemid ndash YAC BAC
bull The host organism that the vector is propagated in ndash Bacteria (bacterial origin of replication) ndash Yeast (yeast origin of replication) ndash Shuttle vector (both origins)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3553
Types of vectors
Vector Target fragment length
Plasmid 0-10 kb (total size up to 15 kb)
Cosmid 10-40 kb
P1 artificial chromosome (PAC) 130-150 kbBacterial artificial chromosome (BAC) About 300 kb
Yeast artificial chromosome (YAC) 200 kb to 2 Mb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3653
What vectors will I be covering
Vectors
bull Plasmid vectorso Cloning vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3753
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3853
Plasmid Vectors
bull Plasmids most commonly used in recombinant DNA technology replicate in E coli
bull They have been engineered to optimize their use as vectors in DNA cloning
ndash To simplify working with plasmids their length is reduced
ndash Most plasmid vectors contain little more than the essential nucleotide
sequences required for their use in DNA cloning
bull Plasmid vectors are usually used as either cloning vectors expression vectorsssDNA vectors or transformation vectors
bull They are not used as library vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3953
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4053
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4253
Basic Characteristics of Cloning Vectors
Basic characteristics
1 Origin of replication (ori )
ndash An origin of replication is a sequence of DNA at which replication is initiated on achromosome plasmid or virus
bull small DNAs a single origin is sufficient ndash Larger DNAs have many origins and DNA replication is initiated at all of them
bull Determines the vector copy number
bull Determines the plasmids compatibility its ability to replicate in conjunction withanother plasmid within the same bacterial cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4353
Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4453
Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4553
Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4653
Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4753
Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4853
Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1753
Classification of Restriction Endonucleases
1 Sticky-end cutter vs blunt-end cutter
2 Frequent cutter vs rare cutter
Recognition sequence Cleavage frequency
4 bp (Alu1 HpaII HhaI) (144) = 1256
5 bp (Hga1) (145 ) = 110246 bp (BamH1 HinD111) (146 ) = 14096
7 bp (PpuM1) (147 ) = 116384
8 bp (Not1)
9 bp (AlwN1)10 bp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1853
983122983141983139983151983149983138983145983150983137983150983156 983140983150983137 983154983141983155983156983154983145983139983156983145983151983150
983141983150983140983151983150983157983139983148983141983137983155983141983155
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1953
Classification of Restriction Endonucleases
3 Ambiguous vs precise cutters
eg BsiE1
CGPuPyCGGCPyPuGC
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2053
Classification of Restriction Endonucleases
4 Isoshizomers
Some enzymes have the same target site (recognition site)
Two subtypes
Same target site ndash different cleavage site
eg Sma1 and Xma1
Same target site - same cleavage site
eg Aat1 and Stu1
BamH1 and Bst1
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2253
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2353
Restriction digestionCommercial restriction enzymes
1 Restriction enzymes are commercially available at 5 units per
microL concentration
2 I unit = 1 unit (U) is the amount if enzyme that catalyses the
conditions
3 Enzymes come in storage buffer containing EDTA DTT BSAand Glycerol
4 Too much enzyme in the reaction mix can cause star activity
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2453
Star activity
Loss of specificity of the restriction enzyme (typeII) underextreme non-standard conditions is called star activity
Under such conditions the restriction enzyme may cleave similarbut non identical target sequences
Non-standard conditions include1 high glycerol concentration gt5 vv
2 gt100 units per ug of DNA
3 Low ionic strength buffer lt25mM (usually is 100-150 mM)
4 high pH (gt 80) usually pH is around 75 presence of organic solvents DMSO ethanol PEGdimethyl acetamide
6 Cations other than Mg++ eg Cu++ Co++Zn++ Mn++
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2553
Restriction digestion
Stopping the Restriction digestion
1 Heat inactivation of restriction enzymes
Place reaction mix in a waterbath set at 65oCNote not all Restriction enzymes can be heat inactivated egPvu1 Bgl II BamH1 Acc1
2 Add 2ul of 25 mM EDTAEDTA chelates the Mg++ and makes it unavailable to theRestriction Endonuclease
3 Phenol-chloroform extraction purificationRemoves all proteins including the enzymes
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2653
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Single Digests
EcoRI BamHI NcoI
Number offragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2753
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
U n d i g e s t e d
N c o I
EcoRI BamHI NcoI
Numberof
fragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2853
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Double Digests
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2953
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
E c o R I
N c o I
U n d i g e s t e d
B a m H I
N c o I
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3053
SummaryRestriction Digestion is the process of cutting DNA molecules into smaller pieces
with special enzymes called Restriction Endonucleases (sometimes just called
Restriction Enzymes or REs) These special enzymes recognize specificsequences in the DNA molecule (for example GATATC) wherever that sequenceoccurs in the DNA
httphigheredmcgraw-hillcomsites9834092339student_view0chapter18restriction_endonucleaseshtml
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3153
Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3253
So what are vectors
bull Vector (molecular biology) vehicle used to transfer genetic material to a target
cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3353
So what are vectors
ndash Plasmid vector
ndash Binary vector a cloning vector used to generate transgenic plants
ndash Cloning vector
ndash Expression vector a plasmid specifically used for protein expression in thetarget
ndash Shuttle vector a vector (usually a plasmid) constructed so that it canpropagate in two different host species
ndash Viral vector a virus modified to deliver foreign genetic material into a cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3453
Vectors can be classified based on
bull The purpose of the vector ndash eg cloning library construction ssDNA production mRNA synthesis expression
multipurpose etc
bull Origin of vector ndash Plasmid ndash Bacteriophage ndash Cosmid ndash Phagemid ndash YAC BAC
bull The host organism that the vector is propagated in ndash Bacteria (bacterial origin of replication) ndash Yeast (yeast origin of replication) ndash Shuttle vector (both origins)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3553
Types of vectors
Vector Target fragment length
Plasmid 0-10 kb (total size up to 15 kb)
Cosmid 10-40 kb
P1 artificial chromosome (PAC) 130-150 kbBacterial artificial chromosome (BAC) About 300 kb
Yeast artificial chromosome (YAC) 200 kb to 2 Mb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3653
What vectors will I be covering
Vectors
bull Plasmid vectorso Cloning vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3753
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3853
Plasmid Vectors
bull Plasmids most commonly used in recombinant DNA technology replicate in E coli
bull They have been engineered to optimize their use as vectors in DNA cloning
ndash To simplify working with plasmids their length is reduced
ndash Most plasmid vectors contain little more than the essential nucleotide
sequences required for their use in DNA cloning
bull Plasmid vectors are usually used as either cloning vectors expression vectorsssDNA vectors or transformation vectors
bull They are not used as library vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3953
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4053
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4253
Basic Characteristics of Cloning Vectors
Basic characteristics
1 Origin of replication (ori )
ndash An origin of replication is a sequence of DNA at which replication is initiated on achromosome plasmid or virus
bull small DNAs a single origin is sufficient ndash Larger DNAs have many origins and DNA replication is initiated at all of them
bull Determines the vector copy number
bull Determines the plasmids compatibility its ability to replicate in conjunction withanother plasmid within the same bacterial cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4353
Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4453
Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4553
Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4653
Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4753
Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4853
Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1853
983122983141983139983151983149983138983145983150983137983150983156 983140983150983137 983154983141983155983156983154983145983139983156983145983151983150
983141983150983140983151983150983157983139983148983141983137983155983141983155
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1953
Classification of Restriction Endonucleases
3 Ambiguous vs precise cutters
eg BsiE1
CGPuPyCGGCPyPuGC
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2053
Classification of Restriction Endonucleases
4 Isoshizomers
Some enzymes have the same target site (recognition site)
Two subtypes
Same target site ndash different cleavage site
eg Sma1 and Xma1
Same target site - same cleavage site
eg Aat1 and Stu1
BamH1 and Bst1
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2253
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2353
Restriction digestionCommercial restriction enzymes
1 Restriction enzymes are commercially available at 5 units per
microL concentration
2 I unit = 1 unit (U) is the amount if enzyme that catalyses the
conditions
3 Enzymes come in storage buffer containing EDTA DTT BSAand Glycerol
4 Too much enzyme in the reaction mix can cause star activity
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2453
Star activity
Loss of specificity of the restriction enzyme (typeII) underextreme non-standard conditions is called star activity
Under such conditions the restriction enzyme may cleave similarbut non identical target sequences
Non-standard conditions include1 high glycerol concentration gt5 vv
2 gt100 units per ug of DNA
3 Low ionic strength buffer lt25mM (usually is 100-150 mM)
4 high pH (gt 80) usually pH is around 75 presence of organic solvents DMSO ethanol PEGdimethyl acetamide
6 Cations other than Mg++ eg Cu++ Co++Zn++ Mn++
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2553
Restriction digestion
Stopping the Restriction digestion
1 Heat inactivation of restriction enzymes
Place reaction mix in a waterbath set at 65oCNote not all Restriction enzymes can be heat inactivated egPvu1 Bgl II BamH1 Acc1
2 Add 2ul of 25 mM EDTAEDTA chelates the Mg++ and makes it unavailable to theRestriction Endonuclease
3 Phenol-chloroform extraction purificationRemoves all proteins including the enzymes
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2653
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Single Digests
EcoRI BamHI NcoI
Number offragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2753
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
U n d i g e s t e d
N c o I
EcoRI BamHI NcoI
Numberof
fragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2853
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Double Digests
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2953
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
E c o R I
N c o I
U n d i g e s t e d
B a m H I
N c o I
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3053
SummaryRestriction Digestion is the process of cutting DNA molecules into smaller pieces
with special enzymes called Restriction Endonucleases (sometimes just called
Restriction Enzymes or REs) These special enzymes recognize specificsequences in the DNA molecule (for example GATATC) wherever that sequenceoccurs in the DNA
httphigheredmcgraw-hillcomsites9834092339student_view0chapter18restriction_endonucleaseshtml
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3153
Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3253
So what are vectors
bull Vector (molecular biology) vehicle used to transfer genetic material to a target
cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3353
So what are vectors
ndash Plasmid vector
ndash Binary vector a cloning vector used to generate transgenic plants
ndash Cloning vector
ndash Expression vector a plasmid specifically used for protein expression in thetarget
ndash Shuttle vector a vector (usually a plasmid) constructed so that it canpropagate in two different host species
ndash Viral vector a virus modified to deliver foreign genetic material into a cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3453
Vectors can be classified based on
bull The purpose of the vector ndash eg cloning library construction ssDNA production mRNA synthesis expression
multipurpose etc
bull Origin of vector ndash Plasmid ndash Bacteriophage ndash Cosmid ndash Phagemid ndash YAC BAC
bull The host organism that the vector is propagated in ndash Bacteria (bacterial origin of replication) ndash Yeast (yeast origin of replication) ndash Shuttle vector (both origins)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3553
Types of vectors
Vector Target fragment length
Plasmid 0-10 kb (total size up to 15 kb)
Cosmid 10-40 kb
P1 artificial chromosome (PAC) 130-150 kbBacterial artificial chromosome (BAC) About 300 kb
Yeast artificial chromosome (YAC) 200 kb to 2 Mb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3653
What vectors will I be covering
Vectors
bull Plasmid vectorso Cloning vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3753
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3853
Plasmid Vectors
bull Plasmids most commonly used in recombinant DNA technology replicate in E coli
bull They have been engineered to optimize their use as vectors in DNA cloning
ndash To simplify working with plasmids their length is reduced
ndash Most plasmid vectors contain little more than the essential nucleotide
sequences required for their use in DNA cloning
bull Plasmid vectors are usually used as either cloning vectors expression vectorsssDNA vectors or transformation vectors
bull They are not used as library vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3953
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4053
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4253
Basic Characteristics of Cloning Vectors
Basic characteristics
1 Origin of replication (ori )
ndash An origin of replication is a sequence of DNA at which replication is initiated on achromosome plasmid or virus
bull small DNAs a single origin is sufficient ndash Larger DNAs have many origins and DNA replication is initiated at all of them
bull Determines the vector copy number
bull Determines the plasmids compatibility its ability to replicate in conjunction withanother plasmid within the same bacterial cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4353
Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4453
Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4553
Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4653
Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4753
Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4853
Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 1953
Classification of Restriction Endonucleases
3 Ambiguous vs precise cutters
eg BsiE1
CGPuPyCGGCPyPuGC
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2053
Classification of Restriction Endonucleases
4 Isoshizomers
Some enzymes have the same target site (recognition site)
Two subtypes
Same target site ndash different cleavage site
eg Sma1 and Xma1
Same target site - same cleavage site
eg Aat1 and Stu1
BamH1 and Bst1
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2253
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2353
Restriction digestionCommercial restriction enzymes
1 Restriction enzymes are commercially available at 5 units per
microL concentration
2 I unit = 1 unit (U) is the amount if enzyme that catalyses the
conditions
3 Enzymes come in storage buffer containing EDTA DTT BSAand Glycerol
4 Too much enzyme in the reaction mix can cause star activity
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2453
Star activity
Loss of specificity of the restriction enzyme (typeII) underextreme non-standard conditions is called star activity
Under such conditions the restriction enzyme may cleave similarbut non identical target sequences
Non-standard conditions include1 high glycerol concentration gt5 vv
2 gt100 units per ug of DNA
3 Low ionic strength buffer lt25mM (usually is 100-150 mM)
4 high pH (gt 80) usually pH is around 75 presence of organic solvents DMSO ethanol PEGdimethyl acetamide
6 Cations other than Mg++ eg Cu++ Co++Zn++ Mn++
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2553
Restriction digestion
Stopping the Restriction digestion
1 Heat inactivation of restriction enzymes
Place reaction mix in a waterbath set at 65oCNote not all Restriction enzymes can be heat inactivated egPvu1 Bgl II BamH1 Acc1
2 Add 2ul of 25 mM EDTAEDTA chelates the Mg++ and makes it unavailable to theRestriction Endonuclease
3 Phenol-chloroform extraction purificationRemoves all proteins including the enzymes
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2653
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Single Digests
EcoRI BamHI NcoI
Number offragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2753
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
U n d i g e s t e d
N c o I
EcoRI BamHI NcoI
Numberof
fragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2853
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Double Digests
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2953
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
E c o R I
N c o I
U n d i g e s t e d
B a m H I
N c o I
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3053
SummaryRestriction Digestion is the process of cutting DNA molecules into smaller pieces
with special enzymes called Restriction Endonucleases (sometimes just called
Restriction Enzymes or REs) These special enzymes recognize specificsequences in the DNA molecule (for example GATATC) wherever that sequenceoccurs in the DNA
httphigheredmcgraw-hillcomsites9834092339student_view0chapter18restriction_endonucleaseshtml
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3153
Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3253
So what are vectors
bull Vector (molecular biology) vehicle used to transfer genetic material to a target
cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3353
So what are vectors
ndash Plasmid vector
ndash Binary vector a cloning vector used to generate transgenic plants
ndash Cloning vector
ndash Expression vector a plasmid specifically used for protein expression in thetarget
ndash Shuttle vector a vector (usually a plasmid) constructed so that it canpropagate in two different host species
ndash Viral vector a virus modified to deliver foreign genetic material into a cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3453
Vectors can be classified based on
bull The purpose of the vector ndash eg cloning library construction ssDNA production mRNA synthesis expression
multipurpose etc
bull Origin of vector ndash Plasmid ndash Bacteriophage ndash Cosmid ndash Phagemid ndash YAC BAC
bull The host organism that the vector is propagated in ndash Bacteria (bacterial origin of replication) ndash Yeast (yeast origin of replication) ndash Shuttle vector (both origins)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3553
Types of vectors
Vector Target fragment length
Plasmid 0-10 kb (total size up to 15 kb)
Cosmid 10-40 kb
P1 artificial chromosome (PAC) 130-150 kbBacterial artificial chromosome (BAC) About 300 kb
Yeast artificial chromosome (YAC) 200 kb to 2 Mb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3653
What vectors will I be covering
Vectors
bull Plasmid vectorso Cloning vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3753
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3853
Plasmid Vectors
bull Plasmids most commonly used in recombinant DNA technology replicate in E coli
bull They have been engineered to optimize their use as vectors in DNA cloning
ndash To simplify working with plasmids their length is reduced
ndash Most plasmid vectors contain little more than the essential nucleotide
sequences required for their use in DNA cloning
bull Plasmid vectors are usually used as either cloning vectors expression vectorsssDNA vectors or transformation vectors
bull They are not used as library vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3953
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4053
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4253
Basic Characteristics of Cloning Vectors
Basic characteristics
1 Origin of replication (ori )
ndash An origin of replication is a sequence of DNA at which replication is initiated on achromosome plasmid or virus
bull small DNAs a single origin is sufficient ndash Larger DNAs have many origins and DNA replication is initiated at all of them
bull Determines the vector copy number
bull Determines the plasmids compatibility its ability to replicate in conjunction withanother plasmid within the same bacterial cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4353
Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4453
Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4553
Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4653
Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4753
Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4853
Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2053
Classification of Restriction Endonucleases
4 Isoshizomers
Some enzymes have the same target site (recognition site)
Two subtypes
Same target site ndash different cleavage site
eg Sma1 and Xma1
Same target site - same cleavage site
eg Aat1 and Stu1
BamH1 and Bst1
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2253
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2353
Restriction digestionCommercial restriction enzymes
1 Restriction enzymes are commercially available at 5 units per
microL concentration
2 I unit = 1 unit (U) is the amount if enzyme that catalyses the
conditions
3 Enzymes come in storage buffer containing EDTA DTT BSAand Glycerol
4 Too much enzyme in the reaction mix can cause star activity
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2453
Star activity
Loss of specificity of the restriction enzyme (typeII) underextreme non-standard conditions is called star activity
Under such conditions the restriction enzyme may cleave similarbut non identical target sequences
Non-standard conditions include1 high glycerol concentration gt5 vv
2 gt100 units per ug of DNA
3 Low ionic strength buffer lt25mM (usually is 100-150 mM)
4 high pH (gt 80) usually pH is around 75 presence of organic solvents DMSO ethanol PEGdimethyl acetamide
6 Cations other than Mg++ eg Cu++ Co++Zn++ Mn++
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2553
Restriction digestion
Stopping the Restriction digestion
1 Heat inactivation of restriction enzymes
Place reaction mix in a waterbath set at 65oCNote not all Restriction enzymes can be heat inactivated egPvu1 Bgl II BamH1 Acc1
2 Add 2ul of 25 mM EDTAEDTA chelates the Mg++ and makes it unavailable to theRestriction Endonuclease
3 Phenol-chloroform extraction purificationRemoves all proteins including the enzymes
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2653
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Single Digests
EcoRI BamHI NcoI
Number offragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2753
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
U n d i g e s t e d
N c o I
EcoRI BamHI NcoI
Numberof
fragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2853
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Double Digests
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2953
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
E c o R I
N c o I
U n d i g e s t e d
B a m H I
N c o I
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3053
SummaryRestriction Digestion is the process of cutting DNA molecules into smaller pieces
with special enzymes called Restriction Endonucleases (sometimes just called
Restriction Enzymes or REs) These special enzymes recognize specificsequences in the DNA molecule (for example GATATC) wherever that sequenceoccurs in the DNA
httphigheredmcgraw-hillcomsites9834092339student_view0chapter18restriction_endonucleaseshtml
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3153
Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3253
So what are vectors
bull Vector (molecular biology) vehicle used to transfer genetic material to a target
cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3353
So what are vectors
ndash Plasmid vector
ndash Binary vector a cloning vector used to generate transgenic plants
ndash Cloning vector
ndash Expression vector a plasmid specifically used for protein expression in thetarget
ndash Shuttle vector a vector (usually a plasmid) constructed so that it canpropagate in two different host species
ndash Viral vector a virus modified to deliver foreign genetic material into a cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3453
Vectors can be classified based on
bull The purpose of the vector ndash eg cloning library construction ssDNA production mRNA synthesis expression
multipurpose etc
bull Origin of vector ndash Plasmid ndash Bacteriophage ndash Cosmid ndash Phagemid ndash YAC BAC
bull The host organism that the vector is propagated in ndash Bacteria (bacterial origin of replication) ndash Yeast (yeast origin of replication) ndash Shuttle vector (both origins)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3553
Types of vectors
Vector Target fragment length
Plasmid 0-10 kb (total size up to 15 kb)
Cosmid 10-40 kb
P1 artificial chromosome (PAC) 130-150 kbBacterial artificial chromosome (BAC) About 300 kb
Yeast artificial chromosome (YAC) 200 kb to 2 Mb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3653
What vectors will I be covering
Vectors
bull Plasmid vectorso Cloning vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3753
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3853
Plasmid Vectors
bull Plasmids most commonly used in recombinant DNA technology replicate in E coli
bull They have been engineered to optimize their use as vectors in DNA cloning
ndash To simplify working with plasmids their length is reduced
ndash Most plasmid vectors contain little more than the essential nucleotide
sequences required for their use in DNA cloning
bull Plasmid vectors are usually used as either cloning vectors expression vectorsssDNA vectors or transformation vectors
bull They are not used as library vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3953
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4053
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4253
Basic Characteristics of Cloning Vectors
Basic characteristics
1 Origin of replication (ori )
ndash An origin of replication is a sequence of DNA at which replication is initiated on achromosome plasmid or virus
bull small DNAs a single origin is sufficient ndash Larger DNAs have many origins and DNA replication is initiated at all of them
bull Determines the vector copy number
bull Determines the plasmids compatibility its ability to replicate in conjunction withanother plasmid within the same bacterial cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4353
Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4453
Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4553
Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4653
Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4753
Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4853
Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2253
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2353
Restriction digestionCommercial restriction enzymes
1 Restriction enzymes are commercially available at 5 units per
microL concentration
2 I unit = 1 unit (U) is the amount if enzyme that catalyses the
conditions
3 Enzymes come in storage buffer containing EDTA DTT BSAand Glycerol
4 Too much enzyme in the reaction mix can cause star activity
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2453
Star activity
Loss of specificity of the restriction enzyme (typeII) underextreme non-standard conditions is called star activity
Under such conditions the restriction enzyme may cleave similarbut non identical target sequences
Non-standard conditions include1 high glycerol concentration gt5 vv
2 gt100 units per ug of DNA
3 Low ionic strength buffer lt25mM (usually is 100-150 mM)
4 high pH (gt 80) usually pH is around 75 presence of organic solvents DMSO ethanol PEGdimethyl acetamide
6 Cations other than Mg++ eg Cu++ Co++Zn++ Mn++
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2553
Restriction digestion
Stopping the Restriction digestion
1 Heat inactivation of restriction enzymes
Place reaction mix in a waterbath set at 65oCNote not all Restriction enzymes can be heat inactivated egPvu1 Bgl II BamH1 Acc1
2 Add 2ul of 25 mM EDTAEDTA chelates the Mg++ and makes it unavailable to theRestriction Endonuclease
3 Phenol-chloroform extraction purificationRemoves all proteins including the enzymes
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2653
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Single Digests
EcoRI BamHI NcoI
Number offragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2753
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
U n d i g e s t e d
N c o I
EcoRI BamHI NcoI
Numberof
fragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2853
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Double Digests
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2953
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
E c o R I
N c o I
U n d i g e s t e d
B a m H I
N c o I
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3053
SummaryRestriction Digestion is the process of cutting DNA molecules into smaller pieces
with special enzymes called Restriction Endonucleases (sometimes just called
Restriction Enzymes or REs) These special enzymes recognize specificsequences in the DNA molecule (for example GATATC) wherever that sequenceoccurs in the DNA
httphigheredmcgraw-hillcomsites9834092339student_view0chapter18restriction_endonucleaseshtml
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3153
Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3253
So what are vectors
bull Vector (molecular biology) vehicle used to transfer genetic material to a target
cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3353
So what are vectors
ndash Plasmid vector
ndash Binary vector a cloning vector used to generate transgenic plants
ndash Cloning vector
ndash Expression vector a plasmid specifically used for protein expression in thetarget
ndash Shuttle vector a vector (usually a plasmid) constructed so that it canpropagate in two different host species
ndash Viral vector a virus modified to deliver foreign genetic material into a cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3453
Vectors can be classified based on
bull The purpose of the vector ndash eg cloning library construction ssDNA production mRNA synthesis expression
multipurpose etc
bull Origin of vector ndash Plasmid ndash Bacteriophage ndash Cosmid ndash Phagemid ndash YAC BAC
bull The host organism that the vector is propagated in ndash Bacteria (bacterial origin of replication) ndash Yeast (yeast origin of replication) ndash Shuttle vector (both origins)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3553
Types of vectors
Vector Target fragment length
Plasmid 0-10 kb (total size up to 15 kb)
Cosmid 10-40 kb
P1 artificial chromosome (PAC) 130-150 kbBacterial artificial chromosome (BAC) About 300 kb
Yeast artificial chromosome (YAC) 200 kb to 2 Mb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3653
What vectors will I be covering
Vectors
bull Plasmid vectorso Cloning vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3753
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3853
Plasmid Vectors
bull Plasmids most commonly used in recombinant DNA technology replicate in E coli
bull They have been engineered to optimize their use as vectors in DNA cloning
ndash To simplify working with plasmids their length is reduced
ndash Most plasmid vectors contain little more than the essential nucleotide
sequences required for their use in DNA cloning
bull Plasmid vectors are usually used as either cloning vectors expression vectorsssDNA vectors or transformation vectors
bull They are not used as library vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3953
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4053
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4253
Basic Characteristics of Cloning Vectors
Basic characteristics
1 Origin of replication (ori )
ndash An origin of replication is a sequence of DNA at which replication is initiated on achromosome plasmid or virus
bull small DNAs a single origin is sufficient ndash Larger DNAs have many origins and DNA replication is initiated at all of them
bull Determines the vector copy number
bull Determines the plasmids compatibility its ability to replicate in conjunction withanother plasmid within the same bacterial cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4353
Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4453
Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4553
Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4653
Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4753
Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4853
Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2253
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2353
Restriction digestionCommercial restriction enzymes
1 Restriction enzymes are commercially available at 5 units per
microL concentration
2 I unit = 1 unit (U) is the amount if enzyme that catalyses the
conditions
3 Enzymes come in storage buffer containing EDTA DTT BSAand Glycerol
4 Too much enzyme in the reaction mix can cause star activity
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2453
Star activity
Loss of specificity of the restriction enzyme (typeII) underextreme non-standard conditions is called star activity
Under such conditions the restriction enzyme may cleave similarbut non identical target sequences
Non-standard conditions include1 high glycerol concentration gt5 vv
2 gt100 units per ug of DNA
3 Low ionic strength buffer lt25mM (usually is 100-150 mM)
4 high pH (gt 80) usually pH is around 75 presence of organic solvents DMSO ethanol PEGdimethyl acetamide
6 Cations other than Mg++ eg Cu++ Co++Zn++ Mn++
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2553
Restriction digestion
Stopping the Restriction digestion
1 Heat inactivation of restriction enzymes
Place reaction mix in a waterbath set at 65oCNote not all Restriction enzymes can be heat inactivated egPvu1 Bgl II BamH1 Acc1
2 Add 2ul of 25 mM EDTAEDTA chelates the Mg++ and makes it unavailable to theRestriction Endonuclease
3 Phenol-chloroform extraction purificationRemoves all proteins including the enzymes
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2653
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Single Digests
EcoRI BamHI NcoI
Number offragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2753
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
U n d i g e s t e d
N c o I
EcoRI BamHI NcoI
Numberof
fragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2853
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Double Digests
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2953
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
E c o R I
N c o I
U n d i g e s t e d
B a m H I
N c o I
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3053
SummaryRestriction Digestion is the process of cutting DNA molecules into smaller pieces
with special enzymes called Restriction Endonucleases (sometimes just called
Restriction Enzymes or REs) These special enzymes recognize specificsequences in the DNA molecule (for example GATATC) wherever that sequenceoccurs in the DNA
httphigheredmcgraw-hillcomsites9834092339student_view0chapter18restriction_endonucleaseshtml
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3153
Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3253
So what are vectors
bull Vector (molecular biology) vehicle used to transfer genetic material to a target
cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3353
So what are vectors
ndash Plasmid vector
ndash Binary vector a cloning vector used to generate transgenic plants
ndash Cloning vector
ndash Expression vector a plasmid specifically used for protein expression in thetarget
ndash Shuttle vector a vector (usually a plasmid) constructed so that it canpropagate in two different host species
ndash Viral vector a virus modified to deliver foreign genetic material into a cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3453
Vectors can be classified based on
bull The purpose of the vector ndash eg cloning library construction ssDNA production mRNA synthesis expression
multipurpose etc
bull Origin of vector ndash Plasmid ndash Bacteriophage ndash Cosmid ndash Phagemid ndash YAC BAC
bull The host organism that the vector is propagated in ndash Bacteria (bacterial origin of replication) ndash Yeast (yeast origin of replication) ndash Shuttle vector (both origins)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3553
Types of vectors
Vector Target fragment length
Plasmid 0-10 kb (total size up to 15 kb)
Cosmid 10-40 kb
P1 artificial chromosome (PAC) 130-150 kbBacterial artificial chromosome (BAC) About 300 kb
Yeast artificial chromosome (YAC) 200 kb to 2 Mb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3653
What vectors will I be covering
Vectors
bull Plasmid vectorso Cloning vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3753
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3853
Plasmid Vectors
bull Plasmids most commonly used in recombinant DNA technology replicate in E coli
bull They have been engineered to optimize their use as vectors in DNA cloning
ndash To simplify working with plasmids their length is reduced
ndash Most plasmid vectors contain little more than the essential nucleotide
sequences required for their use in DNA cloning
bull Plasmid vectors are usually used as either cloning vectors expression vectorsssDNA vectors or transformation vectors
bull They are not used as library vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3953
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4053
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4253
Basic Characteristics of Cloning Vectors
Basic characteristics
1 Origin of replication (ori )
ndash An origin of replication is a sequence of DNA at which replication is initiated on achromosome plasmid or virus
bull small DNAs a single origin is sufficient ndash Larger DNAs have many origins and DNA replication is initiated at all of them
bull Determines the vector copy number
bull Determines the plasmids compatibility its ability to replicate in conjunction withanother plasmid within the same bacterial cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4353
Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4453
Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4553
Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4653
Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4753
Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4853
Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2353
Restriction digestionCommercial restriction enzymes
1 Restriction enzymes are commercially available at 5 units per
microL concentration
2 I unit = 1 unit (U) is the amount if enzyme that catalyses the
conditions
3 Enzymes come in storage buffer containing EDTA DTT BSAand Glycerol
4 Too much enzyme in the reaction mix can cause star activity
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2453
Star activity
Loss of specificity of the restriction enzyme (typeII) underextreme non-standard conditions is called star activity
Under such conditions the restriction enzyme may cleave similarbut non identical target sequences
Non-standard conditions include1 high glycerol concentration gt5 vv
2 gt100 units per ug of DNA
3 Low ionic strength buffer lt25mM (usually is 100-150 mM)
4 high pH (gt 80) usually pH is around 75 presence of organic solvents DMSO ethanol PEGdimethyl acetamide
6 Cations other than Mg++ eg Cu++ Co++Zn++ Mn++
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2553
Restriction digestion
Stopping the Restriction digestion
1 Heat inactivation of restriction enzymes
Place reaction mix in a waterbath set at 65oCNote not all Restriction enzymes can be heat inactivated egPvu1 Bgl II BamH1 Acc1
2 Add 2ul of 25 mM EDTAEDTA chelates the Mg++ and makes it unavailable to theRestriction Endonuclease
3 Phenol-chloroform extraction purificationRemoves all proteins including the enzymes
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2653
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Single Digests
EcoRI BamHI NcoI
Number offragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2753
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
U n d i g e s t e d
N c o I
EcoRI BamHI NcoI
Numberof
fragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2853
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Double Digests
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2953
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
E c o R I
N c o I
U n d i g e s t e d
B a m H I
N c o I
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3053
SummaryRestriction Digestion is the process of cutting DNA molecules into smaller pieces
with special enzymes called Restriction Endonucleases (sometimes just called
Restriction Enzymes or REs) These special enzymes recognize specificsequences in the DNA molecule (for example GATATC) wherever that sequenceoccurs in the DNA
httphigheredmcgraw-hillcomsites9834092339student_view0chapter18restriction_endonucleaseshtml
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3153
Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3253
So what are vectors
bull Vector (molecular biology) vehicle used to transfer genetic material to a target
cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3353
So what are vectors
ndash Plasmid vector
ndash Binary vector a cloning vector used to generate transgenic plants
ndash Cloning vector
ndash Expression vector a plasmid specifically used for protein expression in thetarget
ndash Shuttle vector a vector (usually a plasmid) constructed so that it canpropagate in two different host species
ndash Viral vector a virus modified to deliver foreign genetic material into a cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3453
Vectors can be classified based on
bull The purpose of the vector ndash eg cloning library construction ssDNA production mRNA synthesis expression
multipurpose etc
bull Origin of vector ndash Plasmid ndash Bacteriophage ndash Cosmid ndash Phagemid ndash YAC BAC
bull The host organism that the vector is propagated in ndash Bacteria (bacterial origin of replication) ndash Yeast (yeast origin of replication) ndash Shuttle vector (both origins)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3553
Types of vectors
Vector Target fragment length
Plasmid 0-10 kb (total size up to 15 kb)
Cosmid 10-40 kb
P1 artificial chromosome (PAC) 130-150 kbBacterial artificial chromosome (BAC) About 300 kb
Yeast artificial chromosome (YAC) 200 kb to 2 Mb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3653
What vectors will I be covering
Vectors
bull Plasmid vectorso Cloning vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3753
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3853
Plasmid Vectors
bull Plasmids most commonly used in recombinant DNA technology replicate in E coli
bull They have been engineered to optimize their use as vectors in DNA cloning
ndash To simplify working with plasmids their length is reduced
ndash Most plasmid vectors contain little more than the essential nucleotide
sequences required for their use in DNA cloning
bull Plasmid vectors are usually used as either cloning vectors expression vectorsssDNA vectors or transformation vectors
bull They are not used as library vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3953
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4053
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4253
Basic Characteristics of Cloning Vectors
Basic characteristics
1 Origin of replication (ori )
ndash An origin of replication is a sequence of DNA at which replication is initiated on achromosome plasmid or virus
bull small DNAs a single origin is sufficient ndash Larger DNAs have many origins and DNA replication is initiated at all of them
bull Determines the vector copy number
bull Determines the plasmids compatibility its ability to replicate in conjunction withanother plasmid within the same bacterial cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4353
Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4453
Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4553
Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4653
Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4753
Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4853
Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2453
Star activity
Loss of specificity of the restriction enzyme (typeII) underextreme non-standard conditions is called star activity
Under such conditions the restriction enzyme may cleave similarbut non identical target sequences
Non-standard conditions include1 high glycerol concentration gt5 vv
2 gt100 units per ug of DNA
3 Low ionic strength buffer lt25mM (usually is 100-150 mM)
4 high pH (gt 80) usually pH is around 75 presence of organic solvents DMSO ethanol PEGdimethyl acetamide
6 Cations other than Mg++ eg Cu++ Co++Zn++ Mn++
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2553
Restriction digestion
Stopping the Restriction digestion
1 Heat inactivation of restriction enzymes
Place reaction mix in a waterbath set at 65oCNote not all Restriction enzymes can be heat inactivated egPvu1 Bgl II BamH1 Acc1
2 Add 2ul of 25 mM EDTAEDTA chelates the Mg++ and makes it unavailable to theRestriction Endonuclease
3 Phenol-chloroform extraction purificationRemoves all proteins including the enzymes
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2653
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Single Digests
EcoRI BamHI NcoI
Number offragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2753
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
U n d i g e s t e d
N c o I
EcoRI BamHI NcoI
Numberof
fragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2853
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Double Digests
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2953
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
E c o R I
N c o I
U n d i g e s t e d
B a m H I
N c o I
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3053
SummaryRestriction Digestion is the process of cutting DNA molecules into smaller pieces
with special enzymes called Restriction Endonucleases (sometimes just called
Restriction Enzymes or REs) These special enzymes recognize specificsequences in the DNA molecule (for example GATATC) wherever that sequenceoccurs in the DNA
httphigheredmcgraw-hillcomsites9834092339student_view0chapter18restriction_endonucleaseshtml
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3153
Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3253
So what are vectors
bull Vector (molecular biology) vehicle used to transfer genetic material to a target
cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3353
So what are vectors
ndash Plasmid vector
ndash Binary vector a cloning vector used to generate transgenic plants
ndash Cloning vector
ndash Expression vector a plasmid specifically used for protein expression in thetarget
ndash Shuttle vector a vector (usually a plasmid) constructed so that it canpropagate in two different host species
ndash Viral vector a virus modified to deliver foreign genetic material into a cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3453
Vectors can be classified based on
bull The purpose of the vector ndash eg cloning library construction ssDNA production mRNA synthesis expression
multipurpose etc
bull Origin of vector ndash Plasmid ndash Bacteriophage ndash Cosmid ndash Phagemid ndash YAC BAC
bull The host organism that the vector is propagated in ndash Bacteria (bacterial origin of replication) ndash Yeast (yeast origin of replication) ndash Shuttle vector (both origins)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3553
Types of vectors
Vector Target fragment length
Plasmid 0-10 kb (total size up to 15 kb)
Cosmid 10-40 kb
P1 artificial chromosome (PAC) 130-150 kbBacterial artificial chromosome (BAC) About 300 kb
Yeast artificial chromosome (YAC) 200 kb to 2 Mb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3653
What vectors will I be covering
Vectors
bull Plasmid vectorso Cloning vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3753
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3853
Plasmid Vectors
bull Plasmids most commonly used in recombinant DNA technology replicate in E coli
bull They have been engineered to optimize their use as vectors in DNA cloning
ndash To simplify working with plasmids their length is reduced
ndash Most plasmid vectors contain little more than the essential nucleotide
sequences required for their use in DNA cloning
bull Plasmid vectors are usually used as either cloning vectors expression vectorsssDNA vectors or transformation vectors
bull They are not used as library vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3953
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4053
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4253
Basic Characteristics of Cloning Vectors
Basic characteristics
1 Origin of replication (ori )
ndash An origin of replication is a sequence of DNA at which replication is initiated on achromosome plasmid or virus
bull small DNAs a single origin is sufficient ndash Larger DNAs have many origins and DNA replication is initiated at all of them
bull Determines the vector copy number
bull Determines the plasmids compatibility its ability to replicate in conjunction withanother plasmid within the same bacterial cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4353
Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4453
Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4553
Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4653
Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4753
Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4853
Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2553
Restriction digestion
Stopping the Restriction digestion
1 Heat inactivation of restriction enzymes
Place reaction mix in a waterbath set at 65oCNote not all Restriction enzymes can be heat inactivated egPvu1 Bgl II BamH1 Acc1
2 Add 2ul of 25 mM EDTAEDTA chelates the Mg++ and makes it unavailable to theRestriction Endonuclease
3 Phenol-chloroform extraction purificationRemoves all proteins including the enzymes
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2653
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Single Digests
EcoRI BamHI NcoI
Number offragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2753
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
U n d i g e s t e d
N c o I
EcoRI BamHI NcoI
Numberof
fragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2853
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Double Digests
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2953
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
E c o R I
N c o I
U n d i g e s t e d
B a m H I
N c o I
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3053
SummaryRestriction Digestion is the process of cutting DNA molecules into smaller pieces
with special enzymes called Restriction Endonucleases (sometimes just called
Restriction Enzymes or REs) These special enzymes recognize specificsequences in the DNA molecule (for example GATATC) wherever that sequenceoccurs in the DNA
httphigheredmcgraw-hillcomsites9834092339student_view0chapter18restriction_endonucleaseshtml
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3153
Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3253
So what are vectors
bull Vector (molecular biology) vehicle used to transfer genetic material to a target
cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3353
So what are vectors
ndash Plasmid vector
ndash Binary vector a cloning vector used to generate transgenic plants
ndash Cloning vector
ndash Expression vector a plasmid specifically used for protein expression in thetarget
ndash Shuttle vector a vector (usually a plasmid) constructed so that it canpropagate in two different host species
ndash Viral vector a virus modified to deliver foreign genetic material into a cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3453
Vectors can be classified based on
bull The purpose of the vector ndash eg cloning library construction ssDNA production mRNA synthesis expression
multipurpose etc
bull Origin of vector ndash Plasmid ndash Bacteriophage ndash Cosmid ndash Phagemid ndash YAC BAC
bull The host organism that the vector is propagated in ndash Bacteria (bacterial origin of replication) ndash Yeast (yeast origin of replication) ndash Shuttle vector (both origins)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3553
Types of vectors
Vector Target fragment length
Plasmid 0-10 kb (total size up to 15 kb)
Cosmid 10-40 kb
P1 artificial chromosome (PAC) 130-150 kbBacterial artificial chromosome (BAC) About 300 kb
Yeast artificial chromosome (YAC) 200 kb to 2 Mb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3653
What vectors will I be covering
Vectors
bull Plasmid vectorso Cloning vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3753
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3853
Plasmid Vectors
bull Plasmids most commonly used in recombinant DNA technology replicate in E coli
bull They have been engineered to optimize their use as vectors in DNA cloning
ndash To simplify working with plasmids their length is reduced
ndash Most plasmid vectors contain little more than the essential nucleotide
sequences required for their use in DNA cloning
bull Plasmid vectors are usually used as either cloning vectors expression vectorsssDNA vectors or transformation vectors
bull They are not used as library vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3953
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4053
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4253
Basic Characteristics of Cloning Vectors
Basic characteristics
1 Origin of replication (ori )
ndash An origin of replication is a sequence of DNA at which replication is initiated on achromosome plasmid or virus
bull small DNAs a single origin is sufficient ndash Larger DNAs have many origins and DNA replication is initiated at all of them
bull Determines the vector copy number
bull Determines the plasmids compatibility its ability to replicate in conjunction withanother plasmid within the same bacterial cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4353
Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4453
Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4553
Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4653
Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4753
Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4853
Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2653
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Single Digests
EcoRI BamHI NcoI
Number offragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2753
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
U n d i g e s t e d
N c o I
EcoRI BamHI NcoI
Numberof
fragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2853
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Double Digests
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2953
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
E c o R I
N c o I
U n d i g e s t e d
B a m H I
N c o I
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3053
SummaryRestriction Digestion is the process of cutting DNA molecules into smaller pieces
with special enzymes called Restriction Endonucleases (sometimes just called
Restriction Enzymes or REs) These special enzymes recognize specificsequences in the DNA molecule (for example GATATC) wherever that sequenceoccurs in the DNA
httphigheredmcgraw-hillcomsites9834092339student_view0chapter18restriction_endonucleaseshtml
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3153
Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3253
So what are vectors
bull Vector (molecular biology) vehicle used to transfer genetic material to a target
cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3353
So what are vectors
ndash Plasmid vector
ndash Binary vector a cloning vector used to generate transgenic plants
ndash Cloning vector
ndash Expression vector a plasmid specifically used for protein expression in thetarget
ndash Shuttle vector a vector (usually a plasmid) constructed so that it canpropagate in two different host species
ndash Viral vector a virus modified to deliver foreign genetic material into a cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3453
Vectors can be classified based on
bull The purpose of the vector ndash eg cloning library construction ssDNA production mRNA synthesis expression
multipurpose etc
bull Origin of vector ndash Plasmid ndash Bacteriophage ndash Cosmid ndash Phagemid ndash YAC BAC
bull The host organism that the vector is propagated in ndash Bacteria (bacterial origin of replication) ndash Yeast (yeast origin of replication) ndash Shuttle vector (both origins)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3553
Types of vectors
Vector Target fragment length
Plasmid 0-10 kb (total size up to 15 kb)
Cosmid 10-40 kb
P1 artificial chromosome (PAC) 130-150 kbBacterial artificial chromosome (BAC) About 300 kb
Yeast artificial chromosome (YAC) 200 kb to 2 Mb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3653
What vectors will I be covering
Vectors
bull Plasmid vectorso Cloning vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3753
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3853
Plasmid Vectors
bull Plasmids most commonly used in recombinant DNA technology replicate in E coli
bull They have been engineered to optimize their use as vectors in DNA cloning
ndash To simplify working with plasmids their length is reduced
ndash Most plasmid vectors contain little more than the essential nucleotide
sequences required for their use in DNA cloning
bull Plasmid vectors are usually used as either cloning vectors expression vectorsssDNA vectors or transformation vectors
bull They are not used as library vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3953
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4053
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4253
Basic Characteristics of Cloning Vectors
Basic characteristics
1 Origin of replication (ori )
ndash An origin of replication is a sequence of DNA at which replication is initiated on achromosome plasmid or virus
bull small DNAs a single origin is sufficient ndash Larger DNAs have many origins and DNA replication is initiated at all of them
bull Determines the vector copy number
bull Determines the plasmids compatibility its ability to replicate in conjunction withanother plasmid within the same bacterial cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4353
Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4453
Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4553
Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4653
Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4753
Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4853
Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2753
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
U n d i g e s t e d
N c o I
EcoRI BamHI NcoI
Numberof
fragments
3 2 2
Size offragments
150bp135kpb25kbp
500bp35kbp
3kb1kb
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2853
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Double Digests
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2953
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
E c o R I
N c o I
U n d i g e s t e d
B a m H I
N c o I
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3053
SummaryRestriction Digestion is the process of cutting DNA molecules into smaller pieces
with special enzymes called Restriction Endonucleases (sometimes just called
Restriction Enzymes or REs) These special enzymes recognize specificsequences in the DNA molecule (for example GATATC) wherever that sequenceoccurs in the DNA
httphigheredmcgraw-hillcomsites9834092339student_view0chapter18restriction_endonucleaseshtml
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3153
Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3253
So what are vectors
bull Vector (molecular biology) vehicle used to transfer genetic material to a target
cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3353
So what are vectors
ndash Plasmid vector
ndash Binary vector a cloning vector used to generate transgenic plants
ndash Cloning vector
ndash Expression vector a plasmid specifically used for protein expression in thetarget
ndash Shuttle vector a vector (usually a plasmid) constructed so that it canpropagate in two different host species
ndash Viral vector a virus modified to deliver foreign genetic material into a cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3453
Vectors can be classified based on
bull The purpose of the vector ndash eg cloning library construction ssDNA production mRNA synthesis expression
multipurpose etc
bull Origin of vector ndash Plasmid ndash Bacteriophage ndash Cosmid ndash Phagemid ndash YAC BAC
bull The host organism that the vector is propagated in ndash Bacteria (bacterial origin of replication) ndash Yeast (yeast origin of replication) ndash Shuttle vector (both origins)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3553
Types of vectors
Vector Target fragment length
Plasmid 0-10 kb (total size up to 15 kb)
Cosmid 10-40 kb
P1 artificial chromosome (PAC) 130-150 kbBacterial artificial chromosome (BAC) About 300 kb
Yeast artificial chromosome (YAC) 200 kb to 2 Mb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3653
What vectors will I be covering
Vectors
bull Plasmid vectorso Cloning vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3753
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3853
Plasmid Vectors
bull Plasmids most commonly used in recombinant DNA technology replicate in E coli
bull They have been engineered to optimize their use as vectors in DNA cloning
ndash To simplify working with plasmids their length is reduced
ndash Most plasmid vectors contain little more than the essential nucleotide
sequences required for their use in DNA cloning
bull Plasmid vectors are usually used as either cloning vectors expression vectorsssDNA vectors or transformation vectors
bull They are not used as library vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3953
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4053
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4253
Basic Characteristics of Cloning Vectors
Basic characteristics
1 Origin of replication (ori )
ndash An origin of replication is a sequence of DNA at which replication is initiated on achromosome plasmid or virus
bull small DNAs a single origin is sufficient ndash Larger DNAs have many origins and DNA replication is initiated at all of them
bull Determines the vector copy number
bull Determines the plasmids compatibility its ability to replicate in conjunction withanother plasmid within the same bacterial cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4353
Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4453
Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4553
Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4653
Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4753
Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4853
Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2853
EcoRI (150bp) EcoRI (15kbp) NcoI (3 kbp)BamHI (500bp)
Digestions
Fragment is 4kbp long
Double Digests
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2953
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
E c o R I
N c o I
U n d i g e s t e d
B a m H I
N c o I
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3053
SummaryRestriction Digestion is the process of cutting DNA molecules into smaller pieces
with special enzymes called Restriction Endonucleases (sometimes just called
Restriction Enzymes or REs) These special enzymes recognize specificsequences in the DNA molecule (for example GATATC) wherever that sequenceoccurs in the DNA
httphigheredmcgraw-hillcomsites9834092339student_view0chapter18restriction_endonucleaseshtml
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3153
Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3253
So what are vectors
bull Vector (molecular biology) vehicle used to transfer genetic material to a target
cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3353
So what are vectors
ndash Plasmid vector
ndash Binary vector a cloning vector used to generate transgenic plants
ndash Cloning vector
ndash Expression vector a plasmid specifically used for protein expression in thetarget
ndash Shuttle vector a vector (usually a plasmid) constructed so that it canpropagate in two different host species
ndash Viral vector a virus modified to deliver foreign genetic material into a cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3453
Vectors can be classified based on
bull The purpose of the vector ndash eg cloning library construction ssDNA production mRNA synthesis expression
multipurpose etc
bull Origin of vector ndash Plasmid ndash Bacteriophage ndash Cosmid ndash Phagemid ndash YAC BAC
bull The host organism that the vector is propagated in ndash Bacteria (bacterial origin of replication) ndash Yeast (yeast origin of replication) ndash Shuttle vector (both origins)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3553
Types of vectors
Vector Target fragment length
Plasmid 0-10 kb (total size up to 15 kb)
Cosmid 10-40 kb
P1 artificial chromosome (PAC) 130-150 kbBacterial artificial chromosome (BAC) About 300 kb
Yeast artificial chromosome (YAC) 200 kb to 2 Mb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3653
What vectors will I be covering
Vectors
bull Plasmid vectorso Cloning vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3753
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3853
Plasmid Vectors
bull Plasmids most commonly used in recombinant DNA technology replicate in E coli
bull They have been engineered to optimize their use as vectors in DNA cloning
ndash To simplify working with plasmids their length is reduced
ndash Most plasmid vectors contain little more than the essential nucleotide
sequences required for their use in DNA cloning
bull Plasmid vectors are usually used as either cloning vectors expression vectorsssDNA vectors or transformation vectors
bull They are not used as library vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3953
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4053
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4253
Basic Characteristics of Cloning Vectors
Basic characteristics
1 Origin of replication (ori )
ndash An origin of replication is a sequence of DNA at which replication is initiated on achromosome plasmid or virus
bull small DNAs a single origin is sufficient ndash Larger DNAs have many origins and DNA replication is initiated at all of them
bull Determines the vector copy number
bull Determines the plasmids compatibility its ability to replicate in conjunction withanother plasmid within the same bacterial cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4353
Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4453
Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4553
Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4653
Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4753
Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4853
Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 2953
Digestions
4kbp
L a d d e r
E c o R I
B a m H I
E c o R I
N c o I
U n d i g e s t e d
B a m H I
N c o I
EcoRIBamHI EcoRINcoI BamHINcoI
Number offragments
4 4 3
Size offragments
150bp350bp1kbp
25kbp
150bp135kbp15kpb
1kbp
500bp25kbp1kbp
3kbp
2kbp
1kbp
05kbp
025kbp
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3053
SummaryRestriction Digestion is the process of cutting DNA molecules into smaller pieces
with special enzymes called Restriction Endonucleases (sometimes just called
Restriction Enzymes or REs) These special enzymes recognize specificsequences in the DNA molecule (for example GATATC) wherever that sequenceoccurs in the DNA
httphigheredmcgraw-hillcomsites9834092339student_view0chapter18restriction_endonucleaseshtml
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3153
Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3253
So what are vectors
bull Vector (molecular biology) vehicle used to transfer genetic material to a target
cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3353
So what are vectors
ndash Plasmid vector
ndash Binary vector a cloning vector used to generate transgenic plants
ndash Cloning vector
ndash Expression vector a plasmid specifically used for protein expression in thetarget
ndash Shuttle vector a vector (usually a plasmid) constructed so that it canpropagate in two different host species
ndash Viral vector a virus modified to deliver foreign genetic material into a cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3453
Vectors can be classified based on
bull The purpose of the vector ndash eg cloning library construction ssDNA production mRNA synthesis expression
multipurpose etc
bull Origin of vector ndash Plasmid ndash Bacteriophage ndash Cosmid ndash Phagemid ndash YAC BAC
bull The host organism that the vector is propagated in ndash Bacteria (bacterial origin of replication) ndash Yeast (yeast origin of replication) ndash Shuttle vector (both origins)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3553
Types of vectors
Vector Target fragment length
Plasmid 0-10 kb (total size up to 15 kb)
Cosmid 10-40 kb
P1 artificial chromosome (PAC) 130-150 kbBacterial artificial chromosome (BAC) About 300 kb
Yeast artificial chromosome (YAC) 200 kb to 2 Mb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3653
What vectors will I be covering
Vectors
bull Plasmid vectorso Cloning vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3753
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3853
Plasmid Vectors
bull Plasmids most commonly used in recombinant DNA technology replicate in E coli
bull They have been engineered to optimize their use as vectors in DNA cloning
ndash To simplify working with plasmids their length is reduced
ndash Most plasmid vectors contain little more than the essential nucleotide
sequences required for their use in DNA cloning
bull Plasmid vectors are usually used as either cloning vectors expression vectorsssDNA vectors or transformation vectors
bull They are not used as library vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3953
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4053
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4253
Basic Characteristics of Cloning Vectors
Basic characteristics
1 Origin of replication (ori )
ndash An origin of replication is a sequence of DNA at which replication is initiated on achromosome plasmid or virus
bull small DNAs a single origin is sufficient ndash Larger DNAs have many origins and DNA replication is initiated at all of them
bull Determines the vector copy number
bull Determines the plasmids compatibility its ability to replicate in conjunction withanother plasmid within the same bacterial cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4353
Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4453
Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4553
Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4653
Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4753
Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4853
Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3053
SummaryRestriction Digestion is the process of cutting DNA molecules into smaller pieces
with special enzymes called Restriction Endonucleases (sometimes just called
Restriction Enzymes or REs) These special enzymes recognize specificsequences in the DNA molecule (for example GATATC) wherever that sequenceoccurs in the DNA
httphigheredmcgraw-hillcomsites9834092339student_view0chapter18restriction_endonucleaseshtml
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3153
Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3253
So what are vectors
bull Vector (molecular biology) vehicle used to transfer genetic material to a target
cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3353
So what are vectors
ndash Plasmid vector
ndash Binary vector a cloning vector used to generate transgenic plants
ndash Cloning vector
ndash Expression vector a plasmid specifically used for protein expression in thetarget
ndash Shuttle vector a vector (usually a plasmid) constructed so that it canpropagate in two different host species
ndash Viral vector a virus modified to deliver foreign genetic material into a cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3453
Vectors can be classified based on
bull The purpose of the vector ndash eg cloning library construction ssDNA production mRNA synthesis expression
multipurpose etc
bull Origin of vector ndash Plasmid ndash Bacteriophage ndash Cosmid ndash Phagemid ndash YAC BAC
bull The host organism that the vector is propagated in ndash Bacteria (bacterial origin of replication) ndash Yeast (yeast origin of replication) ndash Shuttle vector (both origins)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3553
Types of vectors
Vector Target fragment length
Plasmid 0-10 kb (total size up to 15 kb)
Cosmid 10-40 kb
P1 artificial chromosome (PAC) 130-150 kbBacterial artificial chromosome (BAC) About 300 kb
Yeast artificial chromosome (YAC) 200 kb to 2 Mb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3653
What vectors will I be covering
Vectors
bull Plasmid vectorso Cloning vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3753
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3853
Plasmid Vectors
bull Plasmids most commonly used in recombinant DNA technology replicate in E coli
bull They have been engineered to optimize their use as vectors in DNA cloning
ndash To simplify working with plasmids their length is reduced
ndash Most plasmid vectors contain little more than the essential nucleotide
sequences required for their use in DNA cloning
bull Plasmid vectors are usually used as either cloning vectors expression vectorsssDNA vectors or transformation vectors
bull They are not used as library vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3953
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4053
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4253
Basic Characteristics of Cloning Vectors
Basic characteristics
1 Origin of replication (ori )
ndash An origin of replication is a sequence of DNA at which replication is initiated on achromosome plasmid or virus
bull small DNAs a single origin is sufficient ndash Larger DNAs have many origins and DNA replication is initiated at all of them
bull Determines the vector copy number
bull Determines the plasmids compatibility its ability to replicate in conjunction withanother plasmid within the same bacterial cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4353
Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4453
Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4553
Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4653
Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4753
Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4853
Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3153
Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3253
So what are vectors
bull Vector (molecular biology) vehicle used to transfer genetic material to a target
cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3353
So what are vectors
ndash Plasmid vector
ndash Binary vector a cloning vector used to generate transgenic plants
ndash Cloning vector
ndash Expression vector a plasmid specifically used for protein expression in thetarget
ndash Shuttle vector a vector (usually a plasmid) constructed so that it canpropagate in two different host species
ndash Viral vector a virus modified to deliver foreign genetic material into a cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3453
Vectors can be classified based on
bull The purpose of the vector ndash eg cloning library construction ssDNA production mRNA synthesis expression
multipurpose etc
bull Origin of vector ndash Plasmid ndash Bacteriophage ndash Cosmid ndash Phagemid ndash YAC BAC
bull The host organism that the vector is propagated in ndash Bacteria (bacterial origin of replication) ndash Yeast (yeast origin of replication) ndash Shuttle vector (both origins)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3553
Types of vectors
Vector Target fragment length
Plasmid 0-10 kb (total size up to 15 kb)
Cosmid 10-40 kb
P1 artificial chromosome (PAC) 130-150 kbBacterial artificial chromosome (BAC) About 300 kb
Yeast artificial chromosome (YAC) 200 kb to 2 Mb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3653
What vectors will I be covering
Vectors
bull Plasmid vectorso Cloning vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3753
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3853
Plasmid Vectors
bull Plasmids most commonly used in recombinant DNA technology replicate in E coli
bull They have been engineered to optimize their use as vectors in DNA cloning
ndash To simplify working with plasmids their length is reduced
ndash Most plasmid vectors contain little more than the essential nucleotide
sequences required for their use in DNA cloning
bull Plasmid vectors are usually used as either cloning vectors expression vectorsssDNA vectors or transformation vectors
bull They are not used as library vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3953
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4053
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4253
Basic Characteristics of Cloning Vectors
Basic characteristics
1 Origin of replication (ori )
ndash An origin of replication is a sequence of DNA at which replication is initiated on achromosome plasmid or virus
bull small DNAs a single origin is sufficient ndash Larger DNAs have many origins and DNA replication is initiated at all of them
bull Determines the vector copy number
bull Determines the plasmids compatibility its ability to replicate in conjunction withanother plasmid within the same bacterial cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4353
Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4453
Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4553
Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4653
Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4753
Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4853
Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3253
So what are vectors
bull Vector (molecular biology) vehicle used to transfer genetic material to a target
cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3353
So what are vectors
ndash Plasmid vector
ndash Binary vector a cloning vector used to generate transgenic plants
ndash Cloning vector
ndash Expression vector a plasmid specifically used for protein expression in thetarget
ndash Shuttle vector a vector (usually a plasmid) constructed so that it canpropagate in two different host species
ndash Viral vector a virus modified to deliver foreign genetic material into a cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3453
Vectors can be classified based on
bull The purpose of the vector ndash eg cloning library construction ssDNA production mRNA synthesis expression
multipurpose etc
bull Origin of vector ndash Plasmid ndash Bacteriophage ndash Cosmid ndash Phagemid ndash YAC BAC
bull The host organism that the vector is propagated in ndash Bacteria (bacterial origin of replication) ndash Yeast (yeast origin of replication) ndash Shuttle vector (both origins)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3553
Types of vectors
Vector Target fragment length
Plasmid 0-10 kb (total size up to 15 kb)
Cosmid 10-40 kb
P1 artificial chromosome (PAC) 130-150 kbBacterial artificial chromosome (BAC) About 300 kb
Yeast artificial chromosome (YAC) 200 kb to 2 Mb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3653
What vectors will I be covering
Vectors
bull Plasmid vectorso Cloning vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3753
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3853
Plasmid Vectors
bull Plasmids most commonly used in recombinant DNA technology replicate in E coli
bull They have been engineered to optimize their use as vectors in DNA cloning
ndash To simplify working with plasmids their length is reduced
ndash Most plasmid vectors contain little more than the essential nucleotide
sequences required for their use in DNA cloning
bull Plasmid vectors are usually used as either cloning vectors expression vectorsssDNA vectors or transformation vectors
bull They are not used as library vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3953
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4053
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4253
Basic Characteristics of Cloning Vectors
Basic characteristics
1 Origin of replication (ori )
ndash An origin of replication is a sequence of DNA at which replication is initiated on achromosome plasmid or virus
bull small DNAs a single origin is sufficient ndash Larger DNAs have many origins and DNA replication is initiated at all of them
bull Determines the vector copy number
bull Determines the plasmids compatibility its ability to replicate in conjunction withanother plasmid within the same bacterial cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4353
Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4453
Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4553
Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4653
Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4753
Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4853
Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3353
So what are vectors
ndash Plasmid vector
ndash Binary vector a cloning vector used to generate transgenic plants
ndash Cloning vector
ndash Expression vector a plasmid specifically used for protein expression in thetarget
ndash Shuttle vector a vector (usually a plasmid) constructed so that it canpropagate in two different host species
ndash Viral vector a virus modified to deliver foreign genetic material into a cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3453
Vectors can be classified based on
bull The purpose of the vector ndash eg cloning library construction ssDNA production mRNA synthesis expression
multipurpose etc
bull Origin of vector ndash Plasmid ndash Bacteriophage ndash Cosmid ndash Phagemid ndash YAC BAC
bull The host organism that the vector is propagated in ndash Bacteria (bacterial origin of replication) ndash Yeast (yeast origin of replication) ndash Shuttle vector (both origins)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3553
Types of vectors
Vector Target fragment length
Plasmid 0-10 kb (total size up to 15 kb)
Cosmid 10-40 kb
P1 artificial chromosome (PAC) 130-150 kbBacterial artificial chromosome (BAC) About 300 kb
Yeast artificial chromosome (YAC) 200 kb to 2 Mb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3653
What vectors will I be covering
Vectors
bull Plasmid vectorso Cloning vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3753
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3853
Plasmid Vectors
bull Plasmids most commonly used in recombinant DNA technology replicate in E coli
bull They have been engineered to optimize their use as vectors in DNA cloning
ndash To simplify working with plasmids their length is reduced
ndash Most plasmid vectors contain little more than the essential nucleotide
sequences required for their use in DNA cloning
bull Plasmid vectors are usually used as either cloning vectors expression vectorsssDNA vectors or transformation vectors
bull They are not used as library vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3953
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4053
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4253
Basic Characteristics of Cloning Vectors
Basic characteristics
1 Origin of replication (ori )
ndash An origin of replication is a sequence of DNA at which replication is initiated on achromosome plasmid or virus
bull small DNAs a single origin is sufficient ndash Larger DNAs have many origins and DNA replication is initiated at all of them
bull Determines the vector copy number
bull Determines the plasmids compatibility its ability to replicate in conjunction withanother plasmid within the same bacterial cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4353
Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4453
Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4553
Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4653
Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4753
Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4853
Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3453
Vectors can be classified based on
bull The purpose of the vector ndash eg cloning library construction ssDNA production mRNA synthesis expression
multipurpose etc
bull Origin of vector ndash Plasmid ndash Bacteriophage ndash Cosmid ndash Phagemid ndash YAC BAC
bull The host organism that the vector is propagated in ndash Bacteria (bacterial origin of replication) ndash Yeast (yeast origin of replication) ndash Shuttle vector (both origins)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3553
Types of vectors
Vector Target fragment length
Plasmid 0-10 kb (total size up to 15 kb)
Cosmid 10-40 kb
P1 artificial chromosome (PAC) 130-150 kbBacterial artificial chromosome (BAC) About 300 kb
Yeast artificial chromosome (YAC) 200 kb to 2 Mb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3653
What vectors will I be covering
Vectors
bull Plasmid vectorso Cloning vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3753
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3853
Plasmid Vectors
bull Plasmids most commonly used in recombinant DNA technology replicate in E coli
bull They have been engineered to optimize their use as vectors in DNA cloning
ndash To simplify working with plasmids their length is reduced
ndash Most plasmid vectors contain little more than the essential nucleotide
sequences required for their use in DNA cloning
bull Plasmid vectors are usually used as either cloning vectors expression vectorsssDNA vectors or transformation vectors
bull They are not used as library vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3953
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4053
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4253
Basic Characteristics of Cloning Vectors
Basic characteristics
1 Origin of replication (ori )
ndash An origin of replication is a sequence of DNA at which replication is initiated on achromosome plasmid or virus
bull small DNAs a single origin is sufficient ndash Larger DNAs have many origins and DNA replication is initiated at all of them
bull Determines the vector copy number
bull Determines the plasmids compatibility its ability to replicate in conjunction withanother plasmid within the same bacterial cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4353
Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4453
Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4553
Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4653
Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4753
Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4853
Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3553
Types of vectors
Vector Target fragment length
Plasmid 0-10 kb (total size up to 15 kb)
Cosmid 10-40 kb
P1 artificial chromosome (PAC) 130-150 kbBacterial artificial chromosome (BAC) About 300 kb
Yeast artificial chromosome (YAC) 200 kb to 2 Mb
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3653
What vectors will I be covering
Vectors
bull Plasmid vectorso Cloning vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3753
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3853
Plasmid Vectors
bull Plasmids most commonly used in recombinant DNA technology replicate in E coli
bull They have been engineered to optimize their use as vectors in DNA cloning
ndash To simplify working with plasmids their length is reduced
ndash Most plasmid vectors contain little more than the essential nucleotide
sequences required for their use in DNA cloning
bull Plasmid vectors are usually used as either cloning vectors expression vectorsssDNA vectors or transformation vectors
bull They are not used as library vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3953
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4053
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4253
Basic Characteristics of Cloning Vectors
Basic characteristics
1 Origin of replication (ori )
ndash An origin of replication is a sequence of DNA at which replication is initiated on achromosome plasmid or virus
bull small DNAs a single origin is sufficient ndash Larger DNAs have many origins and DNA replication is initiated at all of them
bull Determines the vector copy number
bull Determines the plasmids compatibility its ability to replicate in conjunction withanother plasmid within the same bacterial cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4353
Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4453
Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4553
Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4653
Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4753
Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4853
Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3653
What vectors will I be covering
Vectors
bull Plasmid vectorso Cloning vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3753
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3853
Plasmid Vectors
bull Plasmids most commonly used in recombinant DNA technology replicate in E coli
bull They have been engineered to optimize their use as vectors in DNA cloning
ndash To simplify working with plasmids their length is reduced
ndash Most plasmid vectors contain little more than the essential nucleotide
sequences required for their use in DNA cloning
bull Plasmid vectors are usually used as either cloning vectors expression vectorsssDNA vectors or transformation vectors
bull They are not used as library vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3953
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4053
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4253
Basic Characteristics of Cloning Vectors
Basic characteristics
1 Origin of replication (ori )
ndash An origin of replication is a sequence of DNA at which replication is initiated on achromosome plasmid or virus
bull small DNAs a single origin is sufficient ndash Larger DNAs have many origins and DNA replication is initiated at all of them
bull Determines the vector copy number
bull Determines the plasmids compatibility its ability to replicate in conjunction withanother plasmid within the same bacterial cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4353
Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4453
Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4553
Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4653
Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4753
Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4853
Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3753
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3853
Plasmid Vectors
bull Plasmids most commonly used in recombinant DNA technology replicate in E coli
bull They have been engineered to optimize their use as vectors in DNA cloning
ndash To simplify working with plasmids their length is reduced
ndash Most plasmid vectors contain little more than the essential nucleotide
sequences required for their use in DNA cloning
bull Plasmid vectors are usually used as either cloning vectors expression vectorsssDNA vectors or transformation vectors
bull They are not used as library vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3953
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4053
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4253
Basic Characteristics of Cloning Vectors
Basic characteristics
1 Origin of replication (ori )
ndash An origin of replication is a sequence of DNA at which replication is initiated on achromosome plasmid or virus
bull small DNAs a single origin is sufficient ndash Larger DNAs have many origins and DNA replication is initiated at all of them
bull Determines the vector copy number
bull Determines the plasmids compatibility its ability to replicate in conjunction withanother plasmid within the same bacterial cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4353
Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4453
Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4553
Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4653
Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4753
Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4853
Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3853
Plasmid Vectors
bull Plasmids most commonly used in recombinant DNA technology replicate in E coli
bull They have been engineered to optimize their use as vectors in DNA cloning
ndash To simplify working with plasmids their length is reduced
ndash Most plasmid vectors contain little more than the essential nucleotide
sequences required for their use in DNA cloning
bull Plasmid vectors are usually used as either cloning vectors expression vectorsssDNA vectors or transformation vectors
bull They are not used as library vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3953
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4053
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4253
Basic Characteristics of Cloning Vectors
Basic characteristics
1 Origin of replication (ori )
ndash An origin of replication is a sequence of DNA at which replication is initiated on achromosome plasmid or virus
bull small DNAs a single origin is sufficient ndash Larger DNAs have many origins and DNA replication is initiated at all of them
bull Determines the vector copy number
bull Determines the plasmids compatibility its ability to replicate in conjunction withanother plasmid within the same bacterial cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4353
Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4453
Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4553
Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4653
Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4753
Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4853
Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 3953
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4053
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4253
Basic Characteristics of Cloning Vectors
Basic characteristics
1 Origin of replication (ori )
ndash An origin of replication is a sequence of DNA at which replication is initiated on achromosome plasmid or virus
bull small DNAs a single origin is sufficient ndash Larger DNAs have many origins and DNA replication is initiated at all of them
bull Determines the vector copy number
bull Determines the plasmids compatibility its ability to replicate in conjunction withanother plasmid within the same bacterial cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4353
Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4453
Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4553
Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4653
Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4753
Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4853
Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4053
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4253
Basic Characteristics of Cloning Vectors
Basic characteristics
1 Origin of replication (ori )
ndash An origin of replication is a sequence of DNA at which replication is initiated on achromosome plasmid or virus
bull small DNAs a single origin is sufficient ndash Larger DNAs have many origins and DNA replication is initiated at all of them
bull Determines the vector copy number
bull Determines the plasmids compatibility its ability to replicate in conjunction withanother plasmid within the same bacterial cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4353
Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4453
Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4553
Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4653
Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4753
Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4853
Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4153
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4253
Basic Characteristics of Cloning Vectors
Basic characteristics
1 Origin of replication (ori )
ndash An origin of replication is a sequence of DNA at which replication is initiated on achromosome plasmid or virus
bull small DNAs a single origin is sufficient ndash Larger DNAs have many origins and DNA replication is initiated at all of them
bull Determines the vector copy number
bull Determines the plasmids compatibility its ability to replicate in conjunction withanother plasmid within the same bacterial cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4353
Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4453
Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4553
Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4653
Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4753
Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4853
Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4253
Basic Characteristics of Cloning Vectors
Basic characteristics
1 Origin of replication (ori )
ndash An origin of replication is a sequence of DNA at which replication is initiated on achromosome plasmid or virus
bull small DNAs a single origin is sufficient ndash Larger DNAs have many origins and DNA replication is initiated at all of them
bull Determines the vector copy number
bull Determines the plasmids compatibility its ability to replicate in conjunction withanother plasmid within the same bacterial cell
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4353
Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4453
Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4553
Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4653
Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4753
Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4853
Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4353
Basic Characteristics of Cloning Vectors2 Multiple cloning site (MCS)
bull Is a short segment of DNA which contains manybull Restriction sites within an MCS are typically unique occurring only once within a
given plasmid
Usually the is located within a lsquoscorablersquo marker The MCS allows us to cut the
plasmid insert new DNA and re-ligate the scorable marker allows us to see if the
plasmid does indeed have an insert because the insert will disrupt expression of the
marker This is seen in the use of the lac-Z-alpha fragment in bluewhite screening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4453
Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4553
Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4653
Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4753
Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4853
Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4453
Basic Characteristics of Cloning Vectorsbull Selection using selectable markers (antibiotictoxin resistance reporter
genes) or screening using screenable markers (LacZ)
Cloning Vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4553
Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4653
Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4753
Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4853
Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4553
Cloning Vectors
Basic characteristics1 Origin of replication (ori )
2 Multiple cloning site
3 Selection markers
4 Screenable markers
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4653
Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4753
Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4853
Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4653
Cloning Vectors (Plasmid)
pBR322 ampR tetRampR tetR
bull Eg pBR322
Note plasmid nomenclature lower case p for plasmid and then uppercase letters andnumbers that are descriptive B was for Bolivar and R for Rodriguez the surnamesof the folks who created the plasmid
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4753
Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4853
Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4753
Plasmid Cloning Vectors
Plasmid cloning vectors also have the following characteristics
1 Copy Number
2 Stringent vs relaxed plasmid
3 Tem erature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers10 Polycloning site (MCS)
Cosmids
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4853
Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4853
Cosmids(1) a plasmid origin of replication
(2) one or more selectable markers
(3) a number of unique restriction sites where foreign DNA can be inserted
(4) a cos site (a DNA sequence in bacteriophage a that is required for packaging)
Expression vectors
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 4953
Expression vectors
Bacterial Artificial Chromosome (BAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5053
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (YAC)
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5153
Yeast Artificial Chromosome (YAC)
-centromere sequences (CEN)
-Telomere sequences (TEL)
- Autonomous replicating sequences (ARS)for replication in the yeast nucleus
- Ampicillin resistance for propagation in E
coli
-Three markers including a suppressortRNA gene TRP1 and URA3 genes forselection by complementation in theappropriate yeast host cell
Overview
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5253
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted
Basic Characteristics
bull Origin of replication (ori )
bull Multiple cloning site (MCS)bull Selection using selectable markers or screenable markers
Detailed Characteristics
1 Copy Number
2 Stringent vs relaxed plasmid3 Temperature sensitive mutations
4 RNA I and RNA II genes
5 Size of plasmid
6 Par site
7 Cer site
8 Bom site
9 Selectable vs Screenable markers
10 Polycloning site
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
8172019 Restriction Enzymes and Vectors
httpslidepdfcomreaderfullrestriction-enzymes-and-vectors 5353
Next DayTransformation SelectionScreening
