[20] Improved oligonucleotide-directed mutagenesis using M13 vectors

This chapter discusses the improved oligonucleotide-directed mutagenesis using M 13 vectors using E.coli (EcoK/ EcoB) selection system, which provides a convenient way of constructing a series of mutations at high yield.

Abstract

This chapter discusses the improved oligonucleotide-directed mutagenesis using M 13 vectors. An oligonucleotide is synthesized, which is complementary to part of the DNA template but contains an internal mismatch to direct the required change (point mutation, multiple mutation, insertion, or deletion). The simplest approach to mutagenesis in MI3 is “single priming,” where the mutagenic primer is annealed with the single-stranded M13 template and extended with DNA polymerase I. Single-stranded DNA is then prepared from individual plaques and sequenced by the dideoxy method to verify the mutation. In constructing point mutations, often only the required mutation is obtained. However in constructing deletions, usually only a fraction of the hybridization positive clones carry the required mutation. The specificity of priming of the mutagenic oligonucleotide may be checked by using the oligonucleotide in a dideoxy sequencing reaction or in a primer-directed extension reaction followed by restriction endonuclease digestion. The E.coli (EcoK/ EcoB) selection system provides a convenient way of constructing a series of mutations at high yield.