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

Summary 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 ATCG transversion in the nut L region of phage . 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.Abbreviations bp base pairs - RF replicative form - ssDNA single stranded DNA - Ap gene carbenicillin resistance gene - EtBr ethidium bromide - O.D. optical density - Kb kilobases - P_L major leftward promoter of phage