COLD-PCR: a new platform for highly improved mutation detection in cancer and genetic testing

摘要: PCR is widely employed as the initial DNA amplification step for genetic testing and cancer biomarker detection. However, a key limitation of PCR-based methods, including real-time PCR, inability to selectively amplify low levels variant alleles in wild-type allele background. As result, downstream assays are limited their ability identify subtle changes that can have profound impact on clinical decision-making outcome or serve biomarkers. We developed COLD-PCR (co-amplification at lower denaturation temperature-PCR) [Li, Wang, Mamon, Kulke, Berbeco Makrigiorgos (2008) Nat. Med. 14 , 579–584], novel form amplifies minority from mixtures mutation-containing sequences irrespective mutation type position sequence. Consequently, genomic yields products containing high-prevalence be detected. Since constitutes ubiquitous almost all analysis, provides general platform improve sensitivity essentially DNA-variation detection technologies Sanger sequencing, pyrosequencing, single molecule scanning, genotyping methylation assays. combined with new approach boost capabilities existing methods. replaced regular before sequencing detection-sensitivity by up 100-fold identified p53/Kras/EGFR (epidermal growth factor receptor) mutations heterogeneous samples were missed For clinically relevant micro-deletions, enabled exclusive isolation mutants. expected diverse applications fields identification tracing, instability, infectious diseases, prenatal fetal maternal blood.