Simple and efficient method for site-directed mutageneis with double-stranded plasmid DNA

US5633157A · kind A · utility

Assignee

• University of Medicine and Dentistry of New Jersey · US

Inventors

• Sidney Pestka · North Caldwell, US
• Derhsing Lai · Yardley, US
• Xueli Zhu · Iselin, US

Key dates

• Filing date: Feb 15, 1996
• Grant date: May 27, 1997
• Expiry date: Feb 15, 2016

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12N15/102
• WIPO field: Biotechnology
• WIPO sector: Chemistry

Abstract

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.