Oligonucleotide directed mutagenesis: Selection of mutants by hemimethylation of GATC-sequences
Molecular and General Genetics MGGPublished 1 June 1984
Anne Marmenout, E. Remaut, Jacques van Boom, Walter Fiers
Citations12
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TL;DR
A selection procedure for mutants obtained by oligonucleotide directed mutagenesis based on asymmetrical A-methylation of GATC-sequences in the duplex DNA was developed and used to introduce an AT→CG transversion in the nut L region of phage λ.
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 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.
Keywords
Biochemistry, Genetics and Molecular BiologyEnvironmental Science
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