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Simple and efficient method for site-directed mutageneis with double- stranded plasmid DNA
Simple and efficient method for site-directed mutageneis with double- stranded plasmid DNA
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机译:一种简单高效的双链质粒DNA诱变方法
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摘要
This invention relates to a method for preparing site-specific mutations in double-stranded plasmid DNA which comprises the steps of (a) double digesting a plasmid having a site A, which contains a target sequence, and sites B and C, which flank site A, with restriction endonucleases A and B to produce fragment I and with restriction endonucleases A and C to produce fragment II, wherein restriction endonuclease A produces a 3' or 5' overhang; (b) denaturating fragments I and II to form a mixture; (c) reannealing the mixture from step (b) to produce parental homoduplex fragments I and II, heteroduplex fragment III, and heteroduplex fragment IV; (d) extending the 3' ends of the heteroduplex fragments; and (e) ligating the heteroduplex fragments to produce the double-stranded plasmid DNA mutant.
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