南开大学 基因操作原理 第九章.ppt

* * * * * * * * * * * * * * * * * * * * * 3. Site-directed mutagenesis Function: It is very useful to be able to change just one, or a few specific nucleotides in a sequence to test a hypothesis. Methods: (1) The single-primer method (2) Cassette mutagenesis (3) The PCR method of mutagenesis * * Procedure Oligonucleotide with desired mutation is chemically synthesized ssDNA is obtained from cell Inserted in a plasmid The two are hybridized then the oligonucleotide is extended to make dsDNA. DNA synthesis (semi-conservative) produces one normal DNA molecule, one mutant molecule, leading to one normal cell, one mutant cell. (1) The single-primer method * * (2) Cassette mutagenesis This type of mutagenesis involves the cleavage by enzyme (RE) at a site in the plasmid and subsequent ligation of an oligonucleotide containing the mutation in the gene of interest to the plasmid. Usually the RE that cuts at the plasmid and the oligonucleotide is the same, permitting sticky ends of the plasmid and insert to ligate to one another. * * (3) The PCR method of mutagenesis * * * PCR methods of site-directed mutagenesis * * * The long primer method * * * A set of primers contain the desired mutation The mutation target plasmid separated from E.coli After annealing, “cycle extension” is catalyzed by Pfu polymerase Both extension products of up and down primers anneal and format an open plasmid contains mutations. The template DNA is eliminated by DpnI digention with a restriction enzyme specific for methylated DNA. The mutated plasmid is preserved because it was generated in vitro and is therefore Transformation and get the mutated plasmid Dpn I recognition sites: * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Tm Annealing temperature: Tm-5 (℃) prime length: 20 nucleotides G+C=10 A+T=10 (G+C)%=50 a. Tm=(A+T)×2+ (G+C)×4 Tm=10×2+10×4=60 ℃ b. Tm=62.3+0.41 (G+C)%-500/