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

Everett, R.D., and Chambon, P. (1982) A rapid and efficient method for region- and strand-specific mutagenesis of cloned DNA. EMBO Journal, 1 (4). pp. 433-7. ISSN 0261-4189

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Publisher's URL: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC553064/

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.

Item Type:Article
Status:Published
Refereed:Yes
Glasgow Author(s):Everett, Prof Roger
Authors: Everett, R.D., and Chambon, P.
College/School:College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
Journal Name:EMBO Journal
ISSN:0261-4189
ISSN (Online):1460-2075

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