Site-specific mutagenesis using synthetic oligodeoxyribonucleotide primers: I. Optimum conditions and minimum ologodeoxyribonucleotide length. |
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Authors: | S Gillam M Smith |
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Institution: | Department of Biochemistry, Faculty of Medicine, University of British Columbia, 2075 Wesbrook Place, Vancouver, B.C.Canada V6T 1W5 |
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Abstract: | 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. |
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Keywords: | øX174 DNA mismatch mutations transitions transversions DNA polymerase I transfection |
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