A simple and rapid method for the selection of oligodeoxynucleotide-directed mutants |
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Authors: | M A Vandeyar M P Weiner C J Hutton C A Batt |
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Institution: | Department of Food Science, Cornell University, Ithaca, NY 14853. |
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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. |
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