Recombinant DNA & gene cloning Biology Donald Winslow 5 October 2010
Genetic engineering Synthesis of drugs & biological products Transgenic crop plants Transgenic animals Medical diagnosis Medical therapy Personal identification Ethical, legal, environmental, & social questions
Recombinant DNA Genetic engineering Made possible by complementarity...and DNA-binding proteins
Applications of recombinant DNA DNA analysis Protein synthesis Studies of gene function Transgenic organisms DNA sequencing
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
Recombinant DNA concepts Splicing Vector Replication Amplification Clone Donor Recipient
Restriction enzymes (endonucleases) Fragments Sticky ends DNA ligase Hybridization EcoR1 palindrome GAATTC CTTAAG
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
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
Cloning vectors Plasmids (transformation) Bacteriophages (infection) Cosmids (transduction) Artificial chromosomes