Site-directed mutagenesis using gapped-heteroduplex plasmid DNA

Abstract Oligonucleotide-directed site-specific mutagenesis is the most effective and powerful method to create desired mutations at a specific site in a gene. The method involves the use of a short, synthetic oligonucleotide carrying the appropriate mutation, which acts as a primer of in vitro DNA synthesis on a complementary single-stranded circular DNA template. There are two basic methods to create a complementary single-stranded circular DNA template; one utilizes double-stranded plasmid (the plasmid method) and the other utilizes single-stranded M13 or phagemid vectors (the M13 method). The M13 method originally developed by Zoller and Smith (1, 2) has been modified to obtain higher yields of mutants (see Chapters 1 to 3). Today, kits for the M13 method are commercially available with which one may get desired mutations with a yield of higher than 50%. However, one of the drawbacks of this method is that one usually has to redone a target fragment into an M13 vector for mutagenesis. In addition, one has to move the mutated fragment back to the original expression plasmid after mutagenesis. In contrast, the plasmid method allows one to directly use double-stranded plasmid DNA for mutagenesis. Although the yields of a desired mutation are usually lower (3-25%) with the plasmid method than with the M13 method, in practice the yields with the plasmid method are high enough to easily obtain desired mutation by a simple screening method.