Defined transversion mutations at a specific position in DNA using synthetic oligodeoxyribonucleotides as mutagens.
The oligodeoxyribonucleotides, pCCCAGCCTCAA, which is complementary to nucleotides 5274--4284 of bacteriophage phi X174 viral DNA , and pCCCAGCCTAAA, which corresponds to the same sequence with a C leads to A change at the ninth nucleotide, were synthesized enzymatically. The second of these oligonucleotides was used as a primer for E. coli DNA polymerase I, from which the 5'-exonculease has been removed by proteolysis (Klenow enzyme), on wild-type phi X174 viral DNA template. After ligation, this yielded closed circular heteroduplex DNA with a G, A mismatch at nucleotide 5276. Transfection of E. coli spheroplasts with the heteroduplex DNA produced phage mutated at this nucleotide (G leads to T in the viral DNA) with high efficiency (13%). The mutant DNA, which corresponds to the gene B mutant am16, was reverted (T leads to G) by the wild type oligonucleotide with an efficiency of 19%. The nucleotide changes  were established by sequence determination of the mutated viral DNA using the enzymatic terminator method. The production of specific transversion mutations, together with a previous demonstration of specific transition mutations (1), established that short enzymatically synthesized oligodeoxyribonucleotides can be used to induce any class of single nucleotide replacement with high efficiency and thus provide a powerful tool for specific genetic manipulations in circular genomes like that of phi X174.
D010584:Bacteriophage phi X 174 / Q000378:metabolism*; D001483:Base Sequence; D004256:DNA Polymerase I / Q000378:metabolism; D004270:DNA, Circular / Q000378:metabolism; D004279:DNA, Viral / Q000096:biosynthesis / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology*; D009153:Mutagens; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology*; D009841:Oligonucleotides / Q000494:pharmacology*; D013698:Templates, Genetic; D014162:Transfection
D004270:DNA, Circular; D004279:DNA, Viral; D009153:Mutagens; D009838:Oligodeoxyribonucleotides; D009841:Oligonucleotides; D004256:DNA Polymerase I