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1. 发明授权

US5633157A Simple and efficient method for site-directed mutageneis with double-stranded plasmid DNA 失效
标题翻译：用双链质粒DNA进行定点诱变的简单有效的方法
公开(公告)号：US5633157A
公开(公告)日：1997-05-27
申请号：US601698
申请日：1996-02-15
申请人： Sidney Pestka , Derhsing Lai , Xueli Zhu
发明人： Sidney Pestka , Derhsing Lai , Xueli Zhu
IPC分类号： C12N15/10 , C12N15/09 , C12N15/01
CPC分类号： C12N15/102
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.
摘要翻译：本发明涉及一种在双链质粒DNA中制备位点特异性突变的方法，该方法包括以下步骤：（a）双重消化含有靶序列的位点A和位点B和C的步骤 A，用限制性内切核酸酶A和B产生片段I和限制性内切核酸酶A和C以产生片段II，其中限制性内切核酸酶A产生3'或5'突出端; （b）使片段I和II变性以形成混合物; （c）将来自步骤（b）的混合物重新退火以产生亲本同源双链体片段I和II，异源双链体片段III和异源双链体片段IV; （d）延伸异源双链片段的3'末端; 和（e）连接异源双链片段以产生双链质粒DNA突变体。

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