14 - Site-Specific Mutagenesis Using the Polymerase Chain Reaction

Abstract

This chapter reviews some of the major PCR site-specific mutagenesis methods proposed to date. The simplest technique in PCR mutagenesis is to put the desired mutated sequence within one of the oligonucleotides used for amplification. This restricts one to small numbers of base changes from the wild-type template sequence, and the altered bases should not be placed at the extreme 3' end of the oligonucleotide because primer extension will be inhibited. A major limitation of this form of mutagenesis is that the mutated sequence appears near the end of the amplified molecule. An early idea was to append to the 5' end of an oligonucleotide the recognition sequence for an enzyme that cuts distal to the recognition sequence. Another method for “embedding” a mutation inside a PCR product uses inverse PCR to amplify an entire plasmid with oligonucleotides whose 5' ends are immediately adjacent to one another on the plasmid DNA. This method is probably limited to fairly short plasmids, and represents a tradeoff between ease of cloning after PCR and the risk of introducing Taq polymerase errors along the entire length of the plasmid. Other general PCR mutagenesis methods rely on “overlap extension” of the ends of PCR-amplifled DNA strands. Two segments of DNA, one lying “upstream” and the other “downstream” of a desired mutation, are amplified using oligonucleotides that introduce the desired mutation and generate PCR products that overlap by about 20 or more nucleotides in the region of the mutation.