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.
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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: | Articles |
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Status: | Published |
Refereed: | Yes |
Glasgow Author(s) Enlighten ID: | Everett, Professor Roger |
Authors: | Everett, R.D., and Chambon, P. |
College/School: | College of Medical Veterinary and Life Sciences > School of Infection & Immunity |
Journal Name: | EMBO Journal |
ISSN: | 0261-4189 |
ISSN (Online): | 1460-2075 |
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