Increased reliability of selective PCR by using additionally mutated primers and a commercial Taq DNA polymerase enhancer

Mol Biotechnol. 1995 Apr;3(2):166-9. doi: 10.1007/BF02789112.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • DNA / isolation & purification*
  • DNA Primers / genetics
  • DNA-Directed DNA Polymerase / chemistry
  • Drug Resistance, Microbial
  • Genes, pol
  • HIV-1 / drug effects
  • HIV-1 / genetics
  • Humans
  • Molecular Sequence Data
  • Point Mutation*
  • Polymerase Chain Reaction / methods*
  • Reproducibility of Results
  • Taq Polymerase
  • Temperature
  • Zidovudine / pharmacology

Substances

  • DNA Primers
  • Zidovudine
  • DNA
  • Taq Polymerase
  • DNA-Directed DNA Polymerase