Recombinant DNA Genetic Engggineering

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?