Embed Size (px)
Citation preview
1
Recombinant DNA&
Genetic Engineeringg g
Genetic Manipulation: Tools
Kathleen Hill
Associate ProfessorDepartment of Biology
The University of Western Ontario
Tools for Genetic Manipulation
• DNA, RNA, cDNA
• Enzymes– Restriction endonucleases
– Ligases
– Polymerases
• Model organisms
281b Hill C8a
2
Recombinant DNA TechnologyKey Concepts
Two key properties of nucleic acids
Complementary
Two key properties of nucleic acids
5’ 3’
ACGTTGCA
5
3’ 5’
AntiparallelACGTTGCA
281b Hill C8aFig 8-2
Property of Protein:nucleic acid interactions
Recombinant DNA TechnologyKey Concepts
Property of Protein:nucleic acid interactions
Sequence specific binding
281b Hill C8aFig 8-2
3
Properties of Enzymology
Recombinant DNA TechnologyKey Concepts
i i
Cutting
Ligation
PastingRestriction Endonuclease Digestion
281b Hill C8aFig 8-5
Recombinant DNA TechnologyKey Concepts
Properties of DNA replication: polymerization
PCR AmplificationCloningin a host organism in a test tube
281b Hill C8aFig 8-8; 8-9
4
Engineering Recombinant DNA
Source DNA
RestrictionRestrictionEndonucleasecuts DNA
FragmentsJoined:HybridizationFollowed by Ligation
281b Hill C8aFig 8-2
Engineering Recombinant DNA
Three Steps1 C t d t DNA• 1. Cut source and vector DNA
– Restriction Endonuclease Digestion
• 2. Insert source fragment into vector
– Hybridization/Ligation– Hybridization/Ligation
• 3. Put recombinant vector into host
– Transformation281b Hill C8a
5
Engineering Recombinant DNA
Source DNA– Genomic DNA
U ll di t f t• Usually a digest fragment
– cDNA• Generated by reverse transcriptase from mRNA
• Chemically synthesized 281b Hill C8a
Source DNAcDNA
• Generated by reverse transcriptase from
RNAmRNA
• Chemically synthesized
281b Hill C8aFig 8-4
6
Cutting DNA
Engineering Recombinant DNA
• Physical shearing– Random sites
• Enzymatic digestingEndon clease digestion site specific– Endonuclease digestion: site specific
– Restriction Endonucleases
281b Hill C8aFig 8-5;8-6
Engineering Recombinant DNA
EcoRI
Cutting DNACutting DNA
Restriction Endonuclease Digestion• Sequence specific
281b Hill C8aFig 8-5;8-6
7
Engineering Recombinant DNA
• Palindrome (Rotational symmetry)• Cuts
Bl t/fl h Bl t d– Blunt/flush -Blunt ends– Staggered -Single stranded “sticky ends”
•3’ overhang•5’ overhang
281b Hill C8a
Restriction Endonuclease Digestion
281b Hill C8aFig 8-6
8
Engineering Recombinant DNA
Vector DNA– Origin of replication
Capable of independent replication in a– Capable of independent replication in a host
– Capable of incorporating DNA
– DNA sequence contains unique restriction site
281b Hill C8aFig 8-7
Insert source fragmentinto vector
• Hybridization– Sticky ends (complementary and antiparallel)– Salt and temperature
• Ligation– DNA ligase – create phosphodiester bonds
complete covalently joined sugar-phosphate backbones
281b Hill C8aFig 8-7
9
Engineering Recombinant DNA
281b Hill C8aFig 8-5
Recombinant DNA TechnologyKey Concepts
Properties of DNA replication: polymerization
PCR AmplificationCloningin a host organism in a test tube
281b Hill C8a
10
Recombinant Molecules: Cloning
• In a host cellR f i t f kb• Range of sizes up to a few kb
• Bacterial cells• Accessory chromosome:
– cloning vector• Choice of vectorChoice of vector• Entry into cell• Replication in cell• Recovery from cell
281b Hill C8a
Fig 8-9
11
Recombinant Molecules: Cloning Vectors
Plasmids• Small circular• Many copies per cell
281b Hill C8a
• Replicate independently• Convenient restriction sites• Unique (single cut) restriction sites
Fig 8-10
Recombinant Molecules: Cloning Vectors
• Means of identifying the recombinant vector• Means of recovery of recombinant vector• Choice depends on size of insert
281b Hill C8a
12
Vectors
Bacteriophage vectors• Single stranded• Double stranded• Size of insert• Dispensible sequence• Headful packaging limits insert size
281b Hill C8a
Putting recombinant DNA into a host
281b Hill C8aFig 8-28
13
Amplify DNAPCR
• Taq polymerase
• Temperature-resistant DNA polymerase
– Thermus aquaticus•Heat resistant
f kb•Best for <2 kb target
281b HiFig 8-8
Amplify DNA
PCRPCR
Melt
281b HiFig 8-8
14
PCR
Anneal
Extend
281b HiFig 8-8
PCR
281b HiFig 8-8
15
PCR
281b HiFig 8-8
• Exponential increase
PCR
• Need to know some sequence information
• Very sensitive
l f f l d l• Can amplify from limited starting material
281b HiFig 8-8
16
What is the size of the PCR product?
PrimerArtefact
Lane M 1 2 3 4 N P 5 6 7 8 9 10
1.8 kb
Gel electrophoresis pseparates DNA fragments on the basis of size.
281b HiFig 8-17
DNA Diagnostics:Who carries an inversion in the Factor VIII gene?
