Oligonucleotide directed mutagenesis: Selection of mutants by hemimethylation of GATC-sequences

Abstract

We have developed a selection procedure for mutants obtained by oligonucleotide directed mutagenesis based on asymmetrical A-methylation of GATC-sequences in the duplex DNA. The method involves the construction of gapped duplexes of circular single-stranded phage DNA. An oligonucleotide, complementary to part of the gap except for a single mismatch, is hybridized to the gapped duplex DNA and the remaining single stranded regions are filled-in enzymatically. When the template is undermethylated, the yield of mutants is almost, solely dependent on the priming efficiency of the oligonucleotide. The approach was used to introduce an AT→CG transversion in the nut L region of phage λ. Under optimal conditions, about 50–60% of the transformants were of the mutant genotype. Although situated adjacent to a known nut L mutation, the present mutation was phenotypically silent. The possibility of screening for mutants by means of a coupled, easily detectable marker was also investigated.