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oligonucleotide-directed mutagenesis


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A technique that allows a specific mutation to be inserted in a gene at a selected site. An olignucleotide sequence complementary to the segment of interest, but containing an alteration at a selected site, is chemically synthesized. Next this is hybridized to a complementary wild-type target gene contained in a single-stranded phage such as M13. The hybridized oligonucleotide fragment is then used as a primer by DNA polymerase I, which extends the molecule while taking instructions from the wild-type complementary strand. The result is a double helix containing a mutant and a wild-type strand. The heteroduplex is then used to transform bacterial cells. From these colonies, strains that contain the mutant homoduplexes can be recovered and propagated. This procedure is also called site-specified mutagenesis. See Chronology, 1978, Hutchison et al.

Subjects: Genetics and Genomics — Chemistry.


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