Increased reliability of selective PCR by using additionally mutated primers and a commercial Taq DNA polymerase enhancer.
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.
De Milito
A
A
Dipartimento di Biologia Molecolare, Università di Siena, Italy.
Catucci
M
M
Iannelli
F
F
Romano
L
L
Zazzi
M
M
Valensin
P E
PE
D001483:Base Sequence; D004247:DNA / Q000302:isolation & purification*; D017931:DNA Primers / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry; D004352:Drug Resistance, Microbial; D015751:Genes, pol; D015497:HIV-1 / Q000187:drug effects / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D019914:Taq Polymerase; D013696:Temperature; D015215:Zidovudine / Q000494:pharmacology
D017931:DNA Primers; D015215:Zidovudine; D004247:DNA; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase