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.
University of Michigan, Ann Arbor 48109-1065.
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Chong
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Garcia
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