发明名称 |
PROCESS FOR CONDUCTING SITE-DIRECTED MUTAGENESIS |
摘要 |
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.
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申请公布号 |
US5071743(A) |
申请公布日期 |
1991.12.10 |
申请号 |
US19890427703 |
申请日期 |
1989.10.27 |
申请人 |
HER MAJESTY THE QUEEN IN RIGHT OF CANADA, AS REPRESENTED BY THE NATIONAL RESEARCH COUNCIL OF CANADA |
发明人 |
SLILATY, STEVE N.;SHEN, SHI-HSIANG;LEBEL, SUSAN |
分类号 |
C12N15/09;C12N9/38;C12N15/10 |
主分类号 |
C12N15/09 |
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