1. Recombinant DNA & gene cloning Biology Donald Winslow 5 October 2010

2. Genetic engineering Synthesis of drugs & biological products Transgenic crop plants Transgenic animals Medical diagnosis Medical therapy Personal identification Ethical, legal, environmental, & social questions

3. Recombinant DNA Genetic engineering Made possible by complementarity...and DNA-binding proteins

4. Applications of recombinant DNA DNA analysis Protein synthesis Studies of gene function Transgenic organisms DNA sequencing

5. Sources of genes to clone Genomic DNA cDNA  Reverse transcriptase & oligo(dT) primer  NaOH denatures RNA/cDNA hybrid  DNA pol I synthesizes complementary DNA strand Synthesized oligonucleotides

6. Recombinant DNA concepts Splicing Vector Replication Amplification Clone Donor Recipient

7. Restriction enzymes (endonucleases)‏ Fragments Sticky ends DNA ligase Hybridization EcoR1 palindrome GAATTC CTTAAG

8. Amplification by PCR (polymerase chain reaction)‏ Denature DNA Hybridize primers Extend primers w/ Taq polymerase Separate synthesized strands Hybridize primers Extend primers to create unit-length strands Purification

9. Cloning genes with microbial cultures Find appropriate cloning vector Get recombinants into vector Replication, mitosis, amplification Selection of transgenic strains  Resistance to the antibiotic ampicillin Recovering amplified recombinant DNA

10. Cloning vectors Plasmids (transformation)‏ Bacteriophages (infection)‏ Cosmids (transduction)‏ Artificial chromosomes