Simple and efficient method for site-directed mutageneis with do

Details Simple and efficient method for site-directed mutageneis with do Simple and efficient method for site-directed mutageneis with do

1996-02-15

1997-05-27

Elliott, George C.

Chemistry: molecular biology and microbiology

Treatment of micro-organisms or enzymes with electrical or...

Modification of viruses

4351721, C12N 1509, C12N 1501

Patent

active

056331576

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.

REFERENCES:
Hofer et. al. (1989) Gene 84, 153-157.
Inouye et. al. (1987), in "Synthesis & Applications of DNA & RNA", ed. Narang, pp. 181-206, Academic Pr.
Morinaba et. al. (1984), Bio/Technol. 2, 636-639.
Slilaty et. al. (1990) Anal. Biochem. 185, 194-200.

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