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Transcript
Benjamin A. Pierce GENETICS A Conceptual Approach FIFTH EDITION CHAPTER 19 Molecular Genetic Analysis and Biotechnology © 2014 W. H. Freeman and Company Cutting and Joining DNA Fragments • Restriction enzymes: recognizing and cutting DNA at specific nucleotide sequences • Type II restriction enzyme: most useful enzyme • By adding methyl groups to the recognition sequence to protect itself from being digested by its own enzyme in bacteria Cutting and Joining DNA Fragments • Cohesive ends: fragments with short, singlestranded overhanging ends • Blunt ends: even-length ends from both single strands Cloning Genes • Plasmid vectors – Plasmids: circular DNA molecules from bacteria – Insert foreign DNA into plasmid using restriction enzymes – Linkers: synthetic DNA fragments containing restriction sites • Transformation of host cells with plasmids • Screening cells for recombinant plasmids – Selectable markers are used to confirm whether the cells have been transformed or not Cloning Genes • Plasmid vectors – Plasmids: circular DNA molecules from bacteria – Insert foreign DNA into plasmid using restriction enzymes – Linkers: synthetic DNA fragments containing restriction sites • Transformation of host cells with plasmids • Screening cells for recombinant plasmids – Selectable markers are used to confirm whether the cells have been transformed or not Cloning Genes • Other gene vectors: – – – – Cosmids Bacterial Artificial Chromosomes (BACs) Yeast Artificial Chromosome Ti plasmid www.monsanto.com/products/pages/droughtgardhybrids.aspx Knockout Mice • A normal gene of the mouse has been fully disabled • Knock-in mice: a mouse carries an inserted DNA sequence at specific locations Silencing Genes with RNAi • siRNAs • Process called RNA interference (RNAi)
