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Appl Environ Microbiol. 1991 October; 57(10): 2888-2890

Site-specific mutagenesis method which completely excludes wild-type DNA from the transformants.

Department of Genetics, Interferon Sciences, Inc., New Brunswick, New Jersey 08901.

ABSTRACT

A highly efficient site-specific mutagenesis method has been devised to exclude wild-type DNA from incorporation into the transformed cells. Two complementary oligonucleotides, corresponding to a target sequence of a DNA molecule and containing an insertion mutation which created an endonuclease restriction site, were synthesized. By using the wild-type DNA molecule flanked by two restriction sites on each side of the target region as a template, the two oligonucleotide primers were extended, enriched, and isolated. The extended products, in turn, were used as templates in a polymerase chain reaction to obtain a mutagenized double-stranded DNA fragment which was conveniently cloned into plasmids by using the flanking restriction sites. Escherichia coli cells transformed by these plasmids were subject to large-scale analysis. One hundred percent of the transformants examined by colony hybridization, restriction enzyme analysis, and DNA sequencing were found to contain the mutant DNA sequence.

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