title: Detection of BRAF V600E mutation with thyroid tissue using pyrosequencing: comparison with PNA-clamping and real-time PCR.
abstract: We used pyrosequencing, peptide nucleic acid (PNA)-clamping polymerase chain reaction (PCR), and real-time PCR to detect the BRAF V600E mutation and to investigate the prognostic effect of the BRAF V600E mutation in paraffin block specimens from 100 patients diagnosed with papillary thyroid carcinoma. Positive rates of PNA-clamping PCR, real-time PCR, and pyrosequencing were 66%, 70%, and 68%, respectively. Pyrosequencing and PNA-clamping PCR detected mutant type in a 99:1 (wild-type: mutant) DNA concentration, and PNA-clamping PCR detected mutant type in a 99.5:0.5 DNA concentration. Clamping PCR showed higher κ value than real-time PCR (0.729 vs 0.626). The BRAF V600E mutation was associated with an advanced stage of cancer (P = .045) and was found to be associated with poor prognostic factors. This study suggests that pyrosequencing can be as sensitive as real-time PCR and that PNA-clamping PCR is a sensitive and reliable method to detect the BRAF V600E mutation.
authors.affiliation: Department of Laboratory Medicine, Chosun University Medical School, Gwangju, Korea.
authors.forename: Seong-Ho
authors.initials: SH
authors.lastname: Kang
authors.forename: Ju Yeon
authors.initials: JY
authors.lastname: Pyo
authors.forename: Seok-Woo
authors.initials: SW
authors.lastname: Yang
authors.forename: Soon Won
authors.initials: SW
authors.lastname: Hong
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D002277:Carcinoma / Q000175:diagnosis* / Q000235:genetics; D002291:Carcinoma, Papillary; D006801:Humans; D008875:Middle Aged; D009154:Mutation; D020135:Peptide Nucleic Acids; D016133:Polymerase Chain Reaction / Q000379:methods; D011379:Prognosis; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA; D000077273:Thyroid Cancer, Papillary; D013961:Thyroid Gland / Q000473:pathology; D013964:Thyroid Neoplasms / Q000175:diagnosis* / Q000235:genetics
chemical_list: D020135:Peptide Nucleic Acids; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf
keywords: BRAF; Peptide nucleic acid; Pyrosequencing; Quantification; Sensitivity