Oligodeoxynucleotide-directed mutagenesis of Escherichia coli and yeast by simple cotransformation of the primer and template

Abstract

A method of oligodeoxynucleotide-directed mutagenesis is presented which requires no in vitro DNA synthesis. Cotransformation of the synthetic primer and single-stranded template into competent spheroplasts of Escherichia coli or Saccharomyces cerevisiae generates the directed mutation. The desired event is detected genetically or by hybridization screening using the mutagenic oligodeoxynucleotide as a probe. The targeted mutation arises at a frequency of approximately 0.1%. In one extensively studied case in E. coli, involving creation of a 99-bp deletion, this procedure produced many fewer untargeted mutation events than did conventional protocols.