title: [Scanning for KRAS, NRAS, BRAF, and PIK3CA mutations by DNA melting analysis with TaqMan probes].
abstract: Scanning for mutations by DNA melting analysis (DMA) is based on asymmetric PCR followed by the melting of duplexes formed by single-stranded amplicons with TaqMan probes. The method is optimally suited for clinical genetic testing; it is easy to perform, high-throughput, and sensitive. The detection limit of mutant alleles by the DMA method is about 3%, which is much higher than the sensitivity of Sanger sequencing. In addition, the DMA method is realized in a closed-tube format, while 2-h assay is carried out in a single tube without any intermediate or additional procedures thereby minimizing the risk of cross contamination of the samples. The validation of the DMA method was performed by scanning for mutations of clinically significant genes KRAS, NRAS, BRAF, and   PIK3CA in 324 DNA samples from tumors of patients with melanoma, colorectal and lung cancer. DNA was isolated either directly from tumor tissues, or from formalin-fixed paraffin-embedded tumor tissues. The detected mutations were verified by Sanger sequencing. The spectra of mutations identified in each tumor type correspond to the literature data and, thus, validate the use of DMA.
authors.affiliation: Research Institute of Carcinogenesis, Blokhin Russian Cancer Research Center, Moscow, 115478 Russia.
authors.forename: I V
authors.initials: IV
authors.lastname: Botezatu
authors.affiliation: Research Institute of Carcinogenesis, Blokhin Russian Cancer Research Center, Moscow, 115478 Russia.
authors.forename: I O
authors.initials: IO
authors.lastname: Panchuk
authors.affiliation: Research Institute of Carcinogenesis, Blokhin Russian Cancer Research Center, Moscow, 115478 Russia.
authors.forename: A M
authors.initials: AM
authors.lastname: Stroganova
authors.affiliation: Research Institute of Carcinogenesis, Blokhin Russian Cancer Research Center, Moscow, 115478 Russia.
authors.forename: A I
authors.initials: AI
authors.lastname: Senderovich
authors.affiliation: Research Institute of Carcinogenesis, Blokhin Russian Cancer Research Center, Moscow, 115478 Russia.
authors.forename: V N
authors.initials: VN
authors.lastname: Kondratova
authors.affiliation: Research Institute of Carcinogenesis, Blokhin Russian Cancer Research Center, Moscow, 115478 Russia.
authors.forename: V P
authors.initials: VP
authors.lastname: Shelepov
authors.affiliation: Research Institute of Carcinogenesis, Blokhin Russian Cancer Research Center, Moscow, 115478 Russia.|alicht@mail.ru.
authors.forename: A V
authors.initials: AV
authors.lastname: Lichtenstein
mesh_terms: D058534:Class I Phosphatidylinositol 3-Kinases / Q000235:genetics*; D015179:Colorectal Neoplasms / Q000235:genetics; D004252:DNA Mutational Analysis; D020558:GTP Phosphohydrolases / Q000235:genetics*; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics; D008545:Melanoma / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics*; D009154:Mutation; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*
chemical_list: C117307:KRAS protein, human; D008565:Membrane Proteins; D058534:Class I Phosphatidylinositol 3-Kinases; C484760:PIK3CA protein, human; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D020558:GTP Phosphohydrolases; C579846:NRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)
keywords: BRAF; DNA melting; KRAS; NRAS; PCR; PIK3CA; TaqMan probes; multiplex analysis; mutation scanning