title: A Sensitive Peptide Nucleic Acid Probe Assay for Detection of BRAF V600 Mutations in Melanoma.
abstract: Mutated v-Raf murine sarcoma viral oncogene homolog B (BRAF) is an important biomarker for the prediction of therapeutic efficacy of several anticancer drugs. The detection of BRAF mutation faces two challenges: Firstly, there are multiple types of mutations, and secondly, tumor samples usually contain various amounts of wild-type, normal tissues. Here, we describe a newly established method for sensitive detection of multiple types of BRAF V600 mutations in excess wild-type background. The method introduced a fluorophore-tagged peptide nucleic acid (PNA) to serve as both polymerase chain reaction (PCR) clamp and sensor probe, which inhibited the amplification of wild-type templates during PCR and revealed multiple types of mutant signals during melting analysis. We demonstrated the design and optimization process of the method, and applied it in the detection of BRAF mutations in 49 melanoma samples. This PNA probe assay method detected three types of mutations in 17 samples, and was much more sensitive than conventional PCR plus Sanger sequencing.
authors: Graduate Institute of Biomedical Sciences, Chang Gung University, Taoyuan, Taiwan, R.O.C.
authors: Tai-Long
authors: TL
authors: Chen
authors: Division of Hematology-Oncology, Department of Internal Medicine, Chang Gung Memorial Hospital, Taoyuan, Taiwan, R.O.C. College of Medicine, Chang Gung University, Taoyuan, Taiwan, R.O.C.
authors: John Wen-Cheng
authors: JW
authors: Chang
authors: Division of Hematology-Oncology, Department of Internal Medicine, Chang Gung Memorial Hospital, Taoyuan, Taiwan, R.O.C. College of Medicine, Chang Gung University, Taoyuan, Taiwan, R.O.C.
authors: Jia-Juan
authors: JJ
authors: Hsieh
authors: Division of Hematology-Oncology, Department of Internal Medicine, Chang Gung Memorial Hospital, Taoyuan, Taiwan, R.O.C. College of Medicine, Chang Gung University, Taoyuan, Taiwan, R.O.C.
authors: Hsin-Yi
authors: HY
authors: Cheng
authors: Graduate Institute of Biomedical Sciences, Chang Gung University, Taoyuan, Taiwan, R.O.C. Department of Thoracic Medicine, Chang Gung Memorial Hospital, Taoyuan, Taiwan, R.O.C. Department of Medical Biotechnology and Laboratory Science, Chang Gung University, Taoyuan, Taiwan, R.O.C. ccchiou@mail.cgu.edu.tw.
authors: Chiuan-Chian
authors: CC
authors: Chiou
mesh_terms: D045744:Cell Line, Tumor; D003062:Codon; D004252:DNA Mutational Analysis; D005838:Genotype; D006801:Humans; D008545:Melanoma / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D020135:Peptide Nucleic Acids; D016133:Polymerase Chain Reaction; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics* / Q000378:metabolism; D012680:Sensitivity and Specificity
chemical_list: D003062:Codon; D020135:Peptide Nucleic Acids; D048493:Proto-Oncogene Proteins B-raf
keywords: BRAF mutations; melanoma; mutation detection; peptide nucleic acid probe