Solid-Phase In Vitro Mutagenesis Using a Plasmid DNA Template

Oligonucleotide-mediated mutagenesis is a predictable and flexible means of introducing very specific changes into cloned DNA sequences to facilitate study of structure-function relationships or modification of restriction endonuclease restriction sites. Several novel polymerase chain reaction (PCR)-based methods of in vitro mutagenesis have been described (1 –6 ). Site-specific mutations are created by introducing mismatches into the oligonucleotides used to prime the in vitro amplification step. Among these PCR-based mutagenesis systems is splicing by overlap extension (1 –5 ), which involves a two-step PCR, followed by cloning of blunt-end products requiring restriction digestion of the PCR product as well as a ligation step. A modification (7 ) simplifies the removal of excess primers and wild-type template, utilizing biotinylated primers and streptavidin-coated magnetic beads to purify the PCR products. Alternatively, PCR amplification is used to create recombinant plasmid circles with discrete, cohesive single-stranded ends (6 ). This method also requires purification steps to remove excess primers. These circles, formed without the use of restriction enzyme digestion or ligation, provide high yields of mutants. However, since the entire vector sequence must be PCR amplified, this method may be limited to relatively small vectors when using standard Taq polymerase because of both its frequency of misincorporation and difficulties with amplification of larger fragments. The nonspecific mutations often introduced by Taq polymerase during PCR (8 ) necessitate sequencing the entire amplicon.