SITE-SPECIFIC MUTAGENESIS USING SYNTHETIC OLIGODEOXYRIBONUCLEOTIDE PRIMERS .1. OPTIMUM CONDITIONS AND MINIMUM OLIGODEOXYRIBONUCLEOTIDE LENGTH

A synthetic oligodeoxyribonucleotide mismatched at a single nucleotide to a specific complementary site on wild-type circular øX174 DNA can be used to produce a defined point mutation after in vitro incorporation into closed circular duplex DNA by elongation with DNA polymerase and ligation followed by transfection of Escherichia coli (Hutchison et al., 1978; Gillam et al., 1979). The present study is an investigation of the optimum conditions required for the oligodeoxyribonucleotide-primed reaction for production of transition and transversion mutations in øX174 DNA, using the large (Klenow) fragment of E. coli DNA polymerase I. Under optimum conditions up to 39% of the progeny of transfection are the desired mutant and significant mutation is observed using a heptadeoxyribonucleotide. © 1979.