An oligonucleotide-directed, in vitro mutagenesis method using ssDNA and preferential DNA amplification of the mutated strand.
The sequential addition of primers in a PCR enables one to preferentially amplify one of the two strands of a heteroduplex DNA template. This serves as the basis for a novel site-directed mutagenesis technique involving a heteroduplex DNA template that has been generated from a single-stranded, wild-type template and one or more mutagenic oligonucleotides. This preferential PCR method yields a mutation efficiency greater than 90% (consistent with the theoretical estimate for the method). The ability to generate multiple mutants also enables the screening of potential mutants by restriction endonuclease digestion.
Chong
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University of Michigan, Ann Arbor 48109-1065.
Garcia
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GA
D001483:Base Sequence; D004277:DNA, Single-Stranded / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides / Q000235:genetics*; D016133:Polymerase Chain Reaction
D004277:DNA, Single-Stranded; D009841:Oligonucleotides