A simple and rapid method for the selection of oligodeoxynucleotide-directed mutants

Gene. 1988 May 15;65(1):129-33. doi: 10.1016/0378-1119(88)90425-8.

Abstract

We describe an in vitro selection procedure for oligodeoxynucleotide-directed mutagenesis, which produces mutants at frequencies of greater than 90%, facilitating the identification of mutants directly by nucleotide sequencing. The method is based on the selective methylation of the mutant strand by the incorporation of 5-methyl-dCTP. Restriction endonuclease digestion of the resulting hemimethylated DNA with MspI results in the nicking of only the nonmethylated-parental strand. The parental strand is removed by treatment with exonuclease III. The mutants are recovered by transformation of a mcrAB strain of Escherichia coli with the nascent strand.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Coliphages / genetics
  • DNA Restriction Enzymes
  • DNA, Single-Stranded / genetics
  • Escherichia coli / genetics*
  • Methylation
  • Mutation*
  • Oligodeoxyribonucleotides*

Substances

  • DNA, Single-Stranded
  • Oligodeoxyribonucleotides
  • DNA Restriction Enzymes