COLD-PCR: improving the sensitivity of molecular diagnostics assays

Expert Rev Mol Diagn. 2011 Mar;11(2):159-69. doi: 10.1586/erm.10.115.

Abstract

The detection of low-abundance DNA variants or mutations is of particular interest to medical diagnostics, individualized patient treatment and cancer prognosis; however, detection sensitivity for low-abundance variants is a pronounced limitation of most currently available molecular assays. We have recently developed coamplification at lower denaturation temperature-PCR (COLD-PCR) to resolve this limitation. This novel form of PCR selectively amplifies low-abundance DNA variants from mixtures of wild-type and mutant-containing (or variant-containing) sequences, irrespective of the mutation type or position on the amplicon, by using a critical denaturation temperature. The use of a lower denaturation temperature in COLD-PCR results in selective denaturation of amplicons with mutation-containing molecules within wild-type mutant heteroduplexes or with a lower melting temperature. COLD-PCR can be used in lieu of conventional PCR in several molecular applications, thus enriching the mutant fraction and improving the sensitivity of downstream mutation detection by up to 100-fold.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Cold Temperature
  • DNA / analysis
  • Humans
  • Mutation
  • Nucleic Acid Denaturation
  • Pathology, Molecular / methods*
  • Pathology, Molecular / standards*
  • Polymerase Chain Reaction / methods*
  • Polymerase Chain Reaction / standards*
  • Sensitivity and Specificity
  • Tumor Suppressor Protein p53 / genetics

Substances

  • Tumor Suppressor Protein p53
  • DNA