title: Rapid and sensitive detection of KRAS mutation after fast-COLD-PCR enrichment and high-resolution melting analysis.
abstract: KRAS mutations exhibit significant predictive and prognostic value in cancer. Efficient, sensitive, and accurate molecular approaches are required to evaluate KRAS mutation status, even when mutant alleles are restricted to a small portion of a clinical sample, which otherwise contains wild-type alleles. We describe a highly sensitive method to detect KRAS mutations by high-resolution melting (HRM) analysis after mutation enrichment by fast-COLDpolymerase chain reaction (PCR). Using 10 ng of starting DNA and after fast-COLD-PCR of a 76-bp region containing KRAS codons 12 and 13; the amplicons undergo a nested conventional PCR reaction followed by HRM analysis. Samples exhibiting aberrant melting profiles are sequenced to identify mutation type and position. Serial dilution experiments indicate a sensitivity of approximately 0.3% mutant-to-wild type for HRM-based mutation detection and the ability to directly sequence mutation-containing samples. A number of lung adenocarcinoma specimens earlier characterized were screened. Fast-COLD-PCR-HRM analysis correctly identified KRAS mutations and also showed a previously undetected, low-level missense GGT > TTT complex mutation. On account of the short target regions and low requirement of starting DNA, this rapid, cost-effective, and sensitive fast-COLD-PCR-HRM approach is expected to find broad application for detecting low-abundance KRAS mutations in a wide range of clinical specimens.
authors.affiliation: Department of Radiation Oncology, Division of Medical Physics and Biophysics, Harvard Medical School, Boston, MA, USA.
authors.forename: Chen
authors.initials: C
authors.lastname: Song
authors.forename: Coren A
authors.initials: CA
authors.lastname: Milbury
authors.forename: Jin
authors.initials: J
authors.lastname: Li
authors.forename: Pingfang
authors.initials: P
authors.lastname: Liu
authors.forename: Meiping
authors.initials: M
authors.lastname: Zhao
authors.forename: Gerasimos Mike
authors.initials: GM
authors.lastname: Makrigiorgos
mesh_terms: D000230:Adenocarcinoma / Q000473:pathology*; D006801:Humans; D008175:Lung Neoplasms / Q000473:pathology*; D057089:Pathology, Molecular / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D012680:Sensitivity and Specificity; D044366:Transition Temperature; D018631:ras Proteins / Q000235:genetics*
chemical_list: C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins