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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1982 Dec;79(23):7214–7217. doi: 10.1073/pnas.79.23.7214

Local mutagenesis within deletion loops of DNA heteroduplexes.

K W Peden, D Nathans
PMCID: PMC347309  PMID: 6760191

Abstract

An efficient method has been 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 two 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 lacking the enzyme uracil N-glycosylase. Pooled progeny DNA is digested with the two restriction enzymes, whose sites had been mutated in the wild-type plasmid, to eliminate the original deletion mutant DNA. Point mutants with C . G-to-T . A transitions are obtained at high frequency after a second transformation. To test the feasibility of the approach, the tetracycline resistance gene of pBR322 was chosen as the target sequence. It was found that 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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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