Technical Report abstract
Nature Medicine 14, 579 - 584 (2008)
Published online: 13 April 2008 | doi:10.1038/nm1708
Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing
Jin Li1, Lilin Wang1, Harvey Mamon1, Matthew H Kulke2, Ross Berbeco1 & G Mike Makrigiorgos1
PCR is widely employed as the initial DNA amplification step for genetic testing. However, a key limitation of PCR-based methods is the inability to selectively amplify low levels of mutations in a wild-type background. As a result, downstream assays are limited in their ability to identify subtle genetic changes that can have a profound impact in clinical decision-making and outcome. Here we describe co-amplification at lower denaturation temperature PCR (COLD-PCR), a novel form of PCR that amplifies minority alleles selectively from mixtures of wild-type and mutation-containing sequences irrespective of the mutation type or position on the sequence. We replaced regular PCR with COLD-PCR before sequencing or genotyping assays to improve mutation detection sensitivity by up to 100-fold and identified new mutations in the genes encoding p53, KRAS and epidermal growth factor in heterogeneous cancer samples that had been missed by the currently used methods. For clinically relevant microdeletions, COLD-PCR enabled exclusive amplification and isolation of the mutants. COLD-PCR will transform the capabilities of PCR-based genetic testing, including applications in cancer, infectious diseases and prenatal identification of fetal alleles in maternal blood.
- Department of Radiation Oncology, Divisions of Genomic Stability and DNA Repair, Physics and Radiation Therapy, Room JF514, 44 Binney Street, Boston, Massachusetts 02115, USA.
- Department of Medical Oncology, Dana Farber Cancer Institute, Harvard Medical School, Room JF514, 44 Binney Street, Boston, Massachusetts 02115, USA.
Correspondence to: G Mike Makrigiorgos1 e-mail: mmakrigiorgos@partners.org