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Site specific mutagenesis using synthetic oligo deoxy ribo nucleotide primers 1. optimum conditions and minimum oligo deoxy ribo nucleotide length

Site specific mutagenesis using synthetic oligo deoxy ribo nucleotide primers 1. optimum conditions and minimum oligo deoxy ribo nucleotide length

Gene 8(1): 81-98

A synthetic oligodeoxyribonucleotide mismatched at a single nucleotide to a specific complementary site on wild-type circular [phage] .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. 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 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.

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