A reliable selective PCR procedure that combines the use of additionally mutated primers with the specificity-enhancing properties of a commercial preparation (Perfect Match, Stratagene) is described. The human immunodeficiency virus type 1 pol gene point mutations known to confer in vitro resistance to azidothymidine were examined as a model for optimization of the assay. The usual strategy of deliberately introducing an additional mismatch 1 residue from the 3' end in the wild-type and mutant primers did not allow reproducible discrimination between wild-type and mutant target sequences. Addition of minimal amounts of Perfect Match to the same PCR mixtures resulted in a significantly enlarged range of selective annealing temperatures, providing a valuable and cost-effective means for reliable detection of known mutations by selective PCR.
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|Titolo:||Increased reliability of 'selective PCR' using additionally mutated primers and a commercial 'Taq DNA polymerase enhancer'.|
|Citazione:||DE MILITO, A., Catucci, M., Iannelli, F., Romano, L., Zazzi, M., & Valensin, P.E. (1995). Increased reliability of 'selective PCR' using additionally mutated primers and a commercial 'Taq DNA polymerase enhancer'. MOLECULAR BIOTECHNOLOGY, 3, 166-169.|
|Appare nelle tipologie:||1.1 Articolo in rivista|
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