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Restriction endonuclease Haemophilus influemzae Rd; Hamilton Smith GTPyPuAC CAPuPyTG HindII plaque EcoRI Palindrome (rotational symmetry) 5’-GAATTC-3’ 3’-CTTAAG-5’ AATTC G Bacterial lawn G CTTAA Sticky end ; protruding, cohesive, overhang Average length ; 46 = 4,096 bp
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Chap. 4. Molecular cloning methods
- Gene Cloning
What is a CLONE ?
genotype
Monozygotic twin
GTPyPuACCAPuPyTG
Haemophilus influemzae Rd; Hamilton Smith
HindII
EcoRI
5’-GAATTC-3’3’-CTTAAG-5’
GCTTAA
AATTCG
Sticky end ; protruding, cohesive, overhang
Average length ; 46 = 4,096 bp
Palindrome(rotational symmetry)
Restriction endonuclease
Bacterial lawn
plaque
1. Restirction endonuclease 2. ligase
- Use ATP for formation of phosphodiester bond
Sticky end ; 5’-protruding, EcoRI : 3’-protruding, PstIBlunt end; SmaIHeteroschizomer ; SmaI, XmaI cf) isoschizomer
5’-P3’-OH
5’-P
3’-OH
EcoRI
PstI
SmaI
Restriction-modification system
Bacterial lawn
plaque
-CH3Methylases methylate the recognition sites for
restriction endonuclease to protect the host DNA.
The methylation is conserved during replication
process.
Vectorplasmid pBR322
The first letter or plasmid should be noted by lower case “p”.
vector insert
1.Replication origin2.Selection marker ; antibiotic resistance gene3.Multicloning sites (MCS)
pUC19
Ampicillin, X-gal, IPTGβ-galactosidase
Self-ligationClone cloning
- Dephosphorylation ; Alkaline phosphatase - directional cloning ; compatible
LyticLysogenic
Vs. colony
Average insert size ; ~ 15 kb
Bacterial lawnplaque
Disadvantages of plasmid- Size - transformation efficiency
Phage vector
Plaque hybridization
colony hybridizationLibrary, screening
P P PP
Homologous gene
혼성화탐침자가방사법
Probe ;1. Polynucletide probe ; homologous gene stringency; low or high
- temp.- denaturing agent ; - ionic strength
2. Oligonucleotide probe ; 17-mer, 21-mer
- degenerated primer U G U
UGG AUG UUC AAA AAC GA W M F K N Q
Cosmid, YAC, BAC; Average length ?
PhagemidHelper phage
cDNA libraryVs.
Genomic DNA libraryDNA :RNA hybrid
5’3’ polymerization 5’3’ exonuclease 3’ 5’ exo.
Nick translation
Could we directly clone a specific genomic sequence
or cDNA by PCR without screening ?
Kary Mullis
Denaturation (94 oC)Annealing (37-72 oC)Polymerization (72 oC)
Taq polymerase Thermus aquaticusThermal cycler = PCR machine
templateRT-PCR
Mbu-1 is specifically expressed in the mouse brain
A
RT-PCR
hear
tkid
ney
lung
brain
mus
clesp
leen
panc
reas
liver
testi
s
mar
ker
Real-time RT-PCR
Rel
ativ
e le
vel
1.0
0.8
0.6
0.4
0.2 0
B
hear
tkid
ney
lung
brain
mus
clesp
leen
panc
reas liver
testi
s
A
1 5 10 15 20 25 30
Cycle Number
Del
ta R
n
Expression vectors
pUC19
Fusion protein
-Toxic - Inclusion body insoluble aggregates Inducible vector
pTrcHis
Affinity chromatography
cf. heme