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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
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Recombinant DNA TechnologyKey Concepts
Two key properties of nucleic acids
Complementary
Two key properties of nucleic acids
5’ 3’
ACGTTGCA
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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PCR
Anneal
Extend
281b HiFig 8-8
PCR
281b HiFig 8-8
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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
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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?
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