文献_COLD-PCR a new platform for highly improved mutation detection in cancer and genetic testing.pdfVIP

Advances in Nucleic Acid Detection and Quantification 427 COLD-PCR: a new platform for highly improved mutation detection in cancer and genetic testing Jin Li and G. Mike Makrigiorgos 1 Division of Genomic Stability and Division of DNA Repair and Medical Physics and Biophysics, Department of Radiation Oncology, Dana Farber Cancer Institute and Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, U.S.A. Abstract 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, is the inability to g selectively amplify low levels of variant alleles in a wild-type allele background. As a result, downstream r assays are limited in their ability to identify subtle genetic changes that can have a profound impact o. on clinical decision-making and outcome or that can serve as cancer biomarkers. We developed COLD- s PCR (co-amplification at lower denaturation temperature-PCR) [Li, Wang, Mamon, Kulke, Berbeco and n Makrigiorgos (2008) Nat. Med. 14, 579–584], a novel form of PCR that amplifies minority alleles selectively a from mixtures of wild-type and mutation-containing sequences irrespective of the mutation type or position r t on the sequence. Consequently, COLD-PCR amplification from genomic DNA yields PCR products containing c o high-prevalence variant alleles that can be detected. Since PCR constitutes a ubiquitous initial step for almost s all genetic analysis, COLD-PCR provides a genera