Site-specific mutagenesis using oligodeoxyribonucleotides: isolation of a phenotypically silent φX174 mutant, with a specific nucleotide deletion, at very high efficiency

Abstract

A decadeoxyribonucleotide, pAAATCCCTCA, was synthesized to complement nucleotides 2920–2930 of coliphage φX174 viral DNA except that the nucleotide corresponding to position 2925 is deleted. The phage DNA sequence in this region codes for the ribosome-binding site of gene H. The oligodeoxyribonucleotide was integrated into double-stranded 0X174 DNA by using it as a primer for DNA polymerase with wild-type DNA template followed by ligation. The resultant heteroduplex DNA was used to transfect Escherichia coli spheroplasts and progeny bacteriophage were isolated. The synthetic decadeoxyribonucleotide was then used to enrich mutant viral DNA from which nucleotide 2925 had been deleted. After three cycles of enrichment and spheroplast transfection only mutant DNA was detectable. The deletion of nucleotide 2925 from the φX174 genome seriously disrupts a sequence complementary to the 3'-terminus of the 16S rRNA of E. coli and also eliminates a translation termination codon from that sequence. However, the mutant grows normally with no readily perceptible phenotype. Thus, a short synthetic oligodeoxyribonucleotide has been used to construct, and to isolate with 100% efficiency, a mutant for which there is no biological selection procedure.