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recombinant PCR

A protocol for site-directed mutation and construction of a vector containing the mutation. A pair of homologous complementary primers that contain the mutation are used separately with other primers each of which, with its mutagenic primer, bracket a unique restriction site in order to construct two double-stranded oligonucleotides that overlap only over the sequence of the mutagenic primers. These double-stranded DNAs are mixed, denatured and annealed to form some of a hybrid that is double-stranded only in the sequence of the mutagenic primers and single-stranded in the direction that extends towards the other external primer sites. PCR amplification and restriction fills in the full-length DNA and prepares it for insertion into a vector. Higuchi, R. (1990) in PCR Protocols: Rapid amplification of cDNA Ends (Innis, M.A., Gelfand, D.H., Sninsky, J.J. and White, T.J., eds.), pp. 177-183, Academic Press, San Diego

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