Recombinant DNA & Genetic Engineering. Tools for Genetic Manipulation

Transcription

1 Recombinant DNA & Genetic Engineering g Genetic Manipulation: Tools Kathleen Hill Associate Professor Department of Biology The University of Western Ontario Tools for Genetic Manipulation DNA, RNA, cdna Enzymes Restriction endonucleases Ligases Polymerases Model organisms 1

2 Recombinant DNA Technology Key Concepts Two key properties of nucleic acids Fig 8-2 ACGT TGCA Complementary 5 3 ACGT TGCA 3 5 Antiparallel Recombinant DNA Technology Key Concepts Property of Protein:nucleic acid interactions Sequence specific binding Fig 8-2 2

3 Recombinant DNA Technology Key Concepts Properties of Enzymology Cutting Restriction i Endonuclease Digestion Ligation Pasting Fig 8-5 Recombinant DNA Technology Key Concepts Properties of DNA replication: polymerization Cloning in a host organism PCR Amplification in a test tube Fig 8-8; 8-9 3

4 Engineering Recombinant DNA Source DNA Restriction Endonuclease cuts DNA Fragments Joined: Hybridization Followed by Ligation Fig 8-2 Engineering Recombinant DNA Three Steps 1. Cut source and vector DNA Restriction Endonuclease Digestion 2. Insert source fragment into vector Hybridization/Ligation 3. Put recombinant vector into host Transformation 4

5 Engineering Recombinant DNA Genomic DNA Source DNA Usually a digest fragment cdna Generated by reverse transcriptase from mrna Chemically synthesized Source DNA cdna Generated by reverse transcriptase from mrna Chemically synthesized Fig 8-4 5

6 Engineering Recombinant DNA Physical shearing Random sites Cutting DNA Enzymatic digesting Endonuclease digestion: site specific Restriction Endonucleases Fig 8-5;8-6 Engineering Recombinant DNA EcoRI Cutting DNA Restriction Endonuclease Digestion Sequence specific Fig 8-5;8-6 6

7 Engineering Recombinant DNA Palindrome (Rotational symmetry) Cuts Blunt/flush -Blunt ends Staggered -Single stranded sticky ends 3 overhang 5 overhang Restriction Endonuclease Digestion Fig 8-6 7

8 Engineering Recombinant DNA Vector DNA Origin of replication Capable of independent replication in a host Capable of incorporating DNA DNA sequence contains unique restriction site Fig 8-7 Insert source fragment into vector Hybridization Sticky ends (complementary and antiparallel) Salt and temperature Ligation DNA ligase create phosphodiester bonds complete covalently joined sugar-phosphate backbones Fig 8-7 8

9 Engineering Recombinant DNA Fig 8-5 Recombinant DNA Technology Key Concepts Properties of DNA replication: polymerization Cloning in a host organism PCR Amplification in a test tube 9

10 Recombinant Molecules: Cloning In a host cell Range of sizes up to a few kb Bacterial cells Accessory chromosome: cloning vector Choice of vector Entry into cell Replication in cell Recovery from cell Fig

11 Recombinant Molecules: Cloning Vectors Plasmids Small circular Many copies per cell 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 11

12 Vectors Bacteriophage vectors Single stranded Double stranded Size of insert Dispensible sequence Headful packaging limits insert size Putting recombinant DNA into a host Fig

13 Amplify DNA PCR Taq polymerase Temperature-resistant DNA polymerase Thermus aquaticus Heat resistant Best for <2 kb target Fig b Hi Amplify DNA PCR Melt Fig b Hi 13

14 PCR Anneal Extend Fig b Hi PCR Fig b Hi 14

15 PCR Fig b Hi PCR Exponential increase Need to know some sequence information Very sensitive Can amplify lf from limited starting material Fig b Hi 15

16 What is the size of the PCR product? Lane M N P kb Primer Artefact Gel electrophoresis separates DNA fragments on the basis of size. Fig b Hi DNA Diagnostics: Who carries an inversion in the Factor VIII gene? 16