title: Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing.
abstract: 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.
authors.affiliation: Department of Radiation Oncology, Divisions of Genomic Stability and DNA Repair, Physics and Radiation Therapy, Room JF514, 44 Binney Street, Boston, Massachusetts 02115, USA.
authors.forename: Jin
authors.initials: J
authors.lastname: Li
authors.forename: Lilin
authors.initials: L
authors.lastname: Wang
authors.forename: Harvey
authors.initials: H
authors.lastname: Mamon
authors.forename: Matthew H
authors.initials: MH
authors.lastname: Kulke
authors.forename: Ross
authors.initials: R
authors.lastname: Berbeco
authors.forename: G Mike
authors.initials: GM
authors.lastname: Makrigiorgos
mesh_terms: D045744:Cell Line, Tumor; D003080:Cold Temperature; D004252:DNA Mutational Analysis / Q000379:methods; D005838:Genotype; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011518:Proto-Oncogene Proteins / Q000235:genetics; D016283:Proto-Oncogene Proteins p21(ras); D012680:Sensitivity and Specificity; D016159:Tumor Suppressor Protein p53 / Q000235:genetics; D018631:ras Proteins / Q000235:genetics
chemical_list: C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D016159:Tumor Suppressor Protein p53; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins