Local mutagenesis within deletion loops of DNA heteroduplexes.

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Abstract
An efficient method was developed to generate base substitution mutations within deletion loops of DNA heteroduplexes. This method utilizes a heteroduplex formed between a deletion mutant cloned in a plasmid vector and its wild-type counterpart from which 2 restriction sites had been removed from the vector. The heteroduplex is exposed to sodium bisulfite to deaminate cytosine residues in the single-stranded loop, and the mutagenized plasmid DNA is used to transform a strain of bacteria [Escherichia coli] lacking the enzyme uracil N-glycosylase. Pooled progeny DNA is digested with the 2 restriction enzymes, whose sites had been mutated in the wild-type plasmid, to eliminate the original deletion mutant DNA. Point mutants with C.cntdot.G-to-T.cntdot.A transitions are obtained at high frequency after a 2nd transformation. To test the feasibility of the approach, the tetracycline resistance gene of [plasmid] pBR322 was chosen as the target sequence. The proportion of tetracycline-sensitive transformants increased as both the size of the heteroduplex loop and the time of incubation with the mutagen increased and this varied from 20 up to 70%. Nucleotide sequence analysis of several tetracycline-sensitive mutants confirmed that C-to-T transitions had been produced in the segment of DNA corresponding to the deletion loop.