title: Optimized allele-specific real-time PCR assays for the detection of common mutations in KRAS and BRAF.
abstract: Mutations in the oncogenes KRAS and BRAF have been identified as prognostic factors in patients with colorectal diseases and as predictors of negative outcome in epidermal growth factor receptor-targeted therapies. Therefore, accurate mutation detection in both genes, KRAS and BRAF, is of increasing clinical relevance. We aimed at optimizing allele-specific real-time PCR assays for the detection of common mutations in KRAS and the BRAF Val600Glu mutation using allele-specific PCR primers for allelic discrimination and probes (TaqMan) for quantification. Each reaction mix contains a co-amplified internal control to exclude false-negative results. Allele-specific real-time PCR assays were evaluated on plasmid model systems providing a mutation detection limit of 10 copies of mutant DNA in proportions as low as 1% of the total DNA. Furthermore, we analyzed 125 DNA samples prepared from archived, formalin-fixed, paraffin-embedded colorectal carcinomas and compared results with those obtained from direct-sequence analysis. All mutations determined by sequence analysis could be recovered by allele-specific PCR assays. In addition, allele-specific PCR assays clearly identified three additional samples affected by a mutation. We propose these allele-specific real-time PCR assays as a low-cost and fast diagnostic tool for accurate detection of KRAS and BRAF mutations that can be applied to clinical samples.
authors.affiliation: Vorarlberg Institute for Vascular Investigation and Treatment, Feldkirch, Austria.
authors.forename: Alois H
authors.initials: AH
authors.lastname: Lang
authors.forename: Heinz
authors.initials: H
authors.lastname: Drexel
authors.forename: Simone
authors.initials: S
authors.lastname: Geller-Rhomberg
authors.forename: Nicole
authors.initials: N
authors.lastname: Stark
authors.forename: Thomas
authors.initials: T
authors.lastname: Winder
authors.forename: Kathrin
authors.initials: K
authors.lastname: Geiger
authors.forename: Axel
authors.initials: A
authors.lastname: Muendlein
mesh_terms: D015179:Colorectal Neoplasms / Q000235:genetics; D011905:Genes, ras / Q000235:genetics*; D006801:Humans; D025202:Molecular Diagnostic Techniques; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D012680:Sensitivity and Specificity; D013048:Specimen Handling
chemical_list: C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf