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. 1982;1(4):433–437. doi: 10.1002/j.1460-2075.1982.tb01187.x

A rapid and efficient method for region- and strand-specific mutagenesis of cloned DNA.

R D Everett, P Chambon
PMCID: PMC553064  PMID: 6329679

Abstract

The single-stranded viral DNA of an M13 phage recombinant containing the early promoter region of SV40 was hybridized with linear, double-stranded replicative form DNA of a related M13 phage containing a short deletion in the cloned SV40 sequence. The heteroduplexes formed between these DNA molecules contained a short, defined single-stranded region in an otherwise duplex molecule. These heteroduplexes were treated with sodium bisulphite to deaminate exposed unpaired cytosines to uracil residues. The single-stranded region was filled in with DNA polymerase I, which incorporates adenine opposite the mutated uracils, and the DNA then transfected into the M13 host JM103 . Viral DNA from the resultant plaques was used for the rapid dideoxy-DNA sequencing procedure; all of the plaques studied contained point mutations within the desired area. This method allows the very rapid and efficient generation of region-directed point mutants which can be quickly sequenced.

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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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