Oligonucleotide directed mutagenesis: selection of mutants by hemimethylation of GATC-sequences.
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 mut L region of phage lambda. 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.
D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D004262:DNA Restriction Enzymes; D004279:DNA, Viral / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005838:Genotype; D008745:Methylation; D009154:Mutation; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology*; D009841:Oligonucleotides / Q000494:pharmacology*; D010957:Plasmids; D013045:Species Specificity
D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D009841:Oligonucleotides; D004262:DNA Restriction Enzymes