title: COLD-PCR: Applications and Advantages.
abstract: Co-amplification at lower denaturation temperature-based polymerase chain reaction (COLD-PCR) is a single-step amplification method that results in the enhancement of both known and unknown minority alleles during PCR, irrespective of mutation type and position. This method is based on exploitation of the critical temperature, Tc, at which mutation-containing DNA is preferentially melted over wild type. COLD-PCR can be a good strategy for mutation detection in specimens with high nonneoplastic cell content, small specimens in which neoplastic cells are difficult to micro-dissect and therefore enrich, and whenever a mutation is suspected to be present but is undetectable using conventional PCR and sequencing methods. We describe in this chapter our COLD-PCR-based pyrosequencing method for KRAS mutation detection in various clinical samples using DNA extracted from either fresh or fixed paraffin-embedded tissue specimens.
authors: Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Unit 72, 1515 Holcombe Blvd, Houston, TX, 77030, USA. zzuo@mdanderson.org.
authors: Zhuang
authors: Z
authors: Zuo
authors: Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Unit 72, 1515 Holcombe Blvd, Houston, TX, 77030, USA. KJJabbar@mdanderson.org.
authors: Kausar J
authors: KJ
authors: Jabbar
mesh_terms: D004252:DNA Mutational Analysis / Q000379:methods; D006801:Humans; D009154:Mutation; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity; D013696:Temperature; D018631:ras Proteins / Q000235:genetics
chemical_list: D018631:ras Proteins
keywords: Co-amplification at lower denaturation temperature PCR (COLD-PCR); KRAS; Mutation detection; Polymerase chain reaction (PCR)