Process for conducting site-directed mutagenesis

Details Process for conducting site-directed mutagenesis Process for conducting site-directed mutagenesis

1989-10-27

1991-12-10

Wax, Robert A.

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

536 27, 935 77, 935 78, C12Q 168, C07H 1512, C12N 1500

Patent

active

050717430

ABSTRACT:
The present invention relates to an approach for conducting site-directed mutagenesis using double-stranded DNA templates. The approach involves the development of a method for generating structures capable of directing full-length complementary-strand synthesis from double-stranded plasmid DNA. These structures are formed following heat denaturation and cooling of linearized plasmid DNA molecules in the presence of what is referred to as a "closing oligonucleotide". A "closing oligonucleotide" is a single-stranded oligonucleotide consisting of a sequence complementary to either or both free ends of one of the two plasmid DNA strands. The "closing oligonucleotide" therefore functions as an agent for recircularization of a DNA strand and generation of a primer-circular template structure suitable for polymerase-dependent full-length complementary-strand synthesis and ligation into a covalently-closed heteroduplex DNA molecule. When combined with a mutagenic oligonucleotide and uracil-substituted DNA templates, this approach allows site-directed mutagenesis to be performed directly on double-stranded DNA with a mutant formation efficiency of about 50%, a level amenable to rapid screening by DNA sequencing.

REFERENCES:
patent: 4851331 (1989-07-01), Vary et al.
patent: 4885248 (1989-12-01), Ahlquist
Cress et al., Gene, 49 (1986) pp. 9-22.
Dente et al., Nucleic Acids Research, vol. 11, No. 6, (1983) pp. 1645-1655.

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