![]() Similarly, as cancer cells are typically heterogeneous in infiltrating and multifocal cancer types, they will be present at low abundances among an excess of normal cells. Furthermore, the difficulty in discerning low-abundance unknown mutations is especially pronounced in heterogeneous specimens from precancerous or cancerous tissue biopsies, sputum, urine, stool and circulating extracellular DNA released in the blood. The ability to accurately examine this heterogeneity remains difficult. Heterogeneity and mosaicism of mutations within tumors and certain syndromes have been well documented. ![]() Many of these mutations may easily be detected at a late stage as a clonal mutation (present in frequencies as low as 100 −1 in wild-type DNA) however, these variants are much more difficult to identify at an early disease stage when present subclonally (100 −1–100 −3 as a ratio to wild-type), randomly (100 −3–100 −6) and spontaneously (100 −6–100 −8), and especially when the mutation type and position are unknown. A particular variant or mutant may confer a selective advantage (a driver mutation) or may be carried through in a neutral manner by linkage processes (passenger mutation) throughout disease progression, tumor evolution and metastasis. However, clinical and diagnostic applications are often limited by accuracy and sensitivity when clinically significant mutations and minority alleles are present at a low abundance relative to the wild-type component of a clinical specimen. In cancer, molecular profiling and disease staging for prognosis, determining personalized treatment and therapy, the assessment of residual disease post-treatment, and the monitoring of therapy outcome and remission/relapse all require accurate identification of DNA mutations and DNA variants. 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. 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. 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. We have recently developed coamplification at lower denaturation temperature-PCR (COLD-PCR) to resolve this limitation. ![]() 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. ![]()
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