COLD-PCR: improving the sensitivity of molecular diagnostics assays. (March 2011)

• Record Type: Journal Article
• Authors: Milbury, Coren A; Li, Jin; Liu, Pingfang; Makrigiorgos, G Mike
• Is Part Of: Expert review of molecular diagnostics. Volume 11:Number 2(2011)
• Page Start: 159
• Page End: 169
• Publication Date: 2011-03
• Subjects: cancer -- coamplification at lower denaturation temperature -- COLD-PCR -- denaturation temperature -- low-abundance mutations -- variant and mutation enrichment; Molecular diagnosis -- Periodicals; 616.0758205
• DOI: 10.1586/erm.10.115
• Languages: English
• ISSNs: 1473-7159

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.