Induction of multiple replacement mutations by oligonucleotide-directed mutagenesis with extended mismatch primers

Abstract

We have developed a method called oligo-scanning mutagenesis that uses oligonucleotides to mutate up to 12 contiguous bases in a single step. Some advantages of this procedure are that the position and sequence of the replacement mutations are completely specified by the investigator, and combinations of mutations can easily be generated. The technique uses a gapped substrate and the Escherichia coli dam methylation error-correcting mechanism to increase the yield of mutants.