期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1982
卷号：79
期号：23
页码：7214-7217
DOI：10.1073/pnas.79.23.7214
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：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.
