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

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.