Defined transversion mutations at a specific position in DNA using synthetic oligodeoxyribonucleotides as mutagens

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Abstract
The oligodeoxyribonucleotides, pCCCAGCCTCAA, which is complementary to nucleotides 5274–4284 of bacteriophage øX174 viral DNA, and pCCCAGCCTAAA, which corresponds to the same sequence with a C → 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′-exonuclease has been removed by proteolysis (Klenow enzyme), on wild-type øX174 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 → T in the viral DNA) with high efficiency (13%). The mutant DNA, which corresponds to the gene B mutant am l6, was reverted (T → C) 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 øX174.