Optimized Allele-Specific Real-Time PCR Assays for the Detection of Common Mutations in KRAS and BRAF

• Autores: Nicole Stark, Thomas Winder, Kathrin Geiger, Axel Muendlein, Alois H. Lang, Heinz Drexel, Simone Geller Rhomberg
• Localización: The Journal of molecular diagnostics, ISSN 1525-1578, Vol. 13, Nº 1, 2011, págs. 23-28
• Idioma: inglés
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• Resumen

  • 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.