Site-specific mutagenesis using synthetic oligodeoxyribonucleotide primers: I. Optimum conditions and minimum ologodeoxyribonucleotide length.
A synthetic oligodeoxyribonucleotide mismatched at a single nucleotide to a specific complementary site on wild-type circular phi X174 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 phi X174 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.
D010584:Bacteriophage phi X 174 / Q000235:genetics*; D004270:DNA, Circular / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000235:genetics*; D009841:Oligonucleotides / Q000235:genetics*; D014162:Transfection
D004270:DNA, Circular; D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D009841:Oligonucleotides; D004259:DNA-Directed DNA Polymerase