A simple and rapid method for the selection of oligodeoxynucleotide-directed mutants.
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.
Vandeyar
M A
MA
Department of Food Science, Cornell University, Ithaca, NY 14853.
Weiner
M P
MP
Hutton
C J
CJ
Batt
C A
CA
D001483:Base Sequence; D003090:Coliphages / Q000235:genetics; D004262:DNA Restriction Enzymes; D004277:DNA, Single-Stranded / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D008745:Methylation; D009154:Mutation; D009838:Oligodeoxyribonucleotides
D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides; D004262:DNA Restriction Enzymes