Chapter 14 Genetic Recombination and Genetic Engineering The biochemistry and molecular biology department of CMU Section DNA Recombination DNA recombination • Homologous Recombination • Conjugation • Transformation • Transduction • Site-specific Recombination • Transposition §1.1 homologous Recombination 　 • Homologous recombination occurs between identical or nearly identical sequences It is also called general recombination 3´ 5´ endonuclease 5´ (recBCD) 3´ 5´ 3´ 3´ 5´ 5´ 3´ 3´ 5´ 5´ 3´ 3´ 5´ 5´ 3´ DNA invading (recA) 5´ 3´ 3´ 5´ 3´ 5´ 5´ 3´ endonuclease Branch migration 5´ 3´ (recA) 3´ 5´ 3´ 5´ 5´ 3´ 5´ 3´ 3´ 5´ 3´ 5´ 5´ 3´ (recBCD) 5´ 3´ 3´ 5´ 3´ 5´ 5´ 3´ DNA ligase Holiday intermediate Holliday intermediate 5´ 3´ 3´ 5´ 3´ 5´ 5´ 3´ 3´ 5´ 3´ 5´ 3´ 5´ 5´ 3´ 3´ 5´ 5´ 3´ endonuclease endonuclease (ruvC) (ruvC) 3´ 5´ 5´ 3´ 3´ 5´ 5´ 3´ DNA ligase 5´ 3´ 5´ 3´ 5´ 3´ 3´ 5´ splice recombinant 3´ 5´ 3´ 5´ 3´ 5´ 5´ 3´ 3´ 5´ 5´ 3´ DNA ligase 3´ 5´ 5´ 3´ patch recombinant 5´ 3´ 3´ 5´ §1.2 Conjugation • Bacterial Conjugation has been defined as the transmission of genetic information from a donor bacterium to a recipient cell through cell-to-cell contact Conjugation process 　 Conjugation process 　 Ligation of blunt ends 　 The addition of a homopolymer tail Artificial linker 　 Adding a sequence of DNA fragment, which contains the cleavage site for restriction endonuclease Artificial linker §2.4 Introduction of recombinant DNA into recipient cell 　 • Introduction: transformation transfection infection Recipient cells • Safe host bacteria • Endonuclease and recombinase deficiency ã Competent cells Đ2.5 Screening for recombinant direct selection • Screen of antibiotic resistance markers • Marker rescue (Insertion inactivation) • In situ hybridization and autoradiography Antibiotic resistance genes 　 The procedure to form recombinant DNA 　 direct selection Screen of antibiotic resistance markers Marker rescue In situ hybridization and autoradiography 　 §2.6 Expression of the cloned gene 　 An expression vector is similar to cloning vectors, but with a major difference: the expression vector must contain a promoter so that proteins can be expressed Expression vector Gene expression include: • eukaryotic expression • prokaryotic expression ...Section DNA Recombination DNA recombination • Homologous Recombination • Conjugation • Transformation • Transduction • Site-specific Recombination • Transposition §1.1 homologous Recombination. .. Transduction 　 　 Đ1.5 Site-specific Recombination ã Site-specific recombination occurs at a specific DNA sequence • The first example was found in the integration between λ DNA and E coli DNA λDNA integration... rH1 ìốảụằựềũ P1 H1 H1 flagellin Recombination signal sequence (RSS) CACAGTG (12/23) ACAAAAACC GTGTCAC TGTTTTTGG RSS Recombination activating gene enzyme (RAG1 and RAG2) §1.6 Transposition • Transposition