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

A method for preparing site-specific mutations in double-stranded plasmid DNA which comprises the steps of:

• (a) double digesting a plasmid having a restriction site A, which contains a target sequence, and restriction sites B and C, which flank restriction 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' overhang;
• (b) denaturating fragments I and II to form a mixture;
• (c) annealing the mixture from step (b) to produce parental homoduplex fragments I and II having a 3'-overhang and a 5'-overhang, heteroduplex fragment III, and heteroduplex fragment IV;
• (d) providing an oligonucleotide primer that can hybridize with the single-stranded region of the heteroduplex fragment III and that has a sequence that directs mutagenesis;
• (e) extending the 3' ends of heteroduplex fragment III;
• and
• (f) ligating heteroduplex fragment III to produce the double-stranded plasmid DNA mutant.