Duplex reverse-hybridization assay for the simultaneous detection of KRAS/BRAF mutations in FFPE-extracted genomic DNA from colorectal cancer specimens

Article type: Research Article

Authors: Buxhofer-Ausch, Veronika^; | Ausch, Christoph^; | Zeillinger, Robert | Oberkanins, Christian | Dandachi, Nadia | Reiner-Concin, Angelika | Kriegshäuser, Gernot^;

Affiliations: Second Department of Medicine, Donauspital, Vienna, Austria | Ludwig Boltzmann Research Society – Cluster Translational Oncology, Vienna, Austria | Department of Surgery, Donauspital, Vienna, Austria | ViennaLab Diagnostics GmbH, Vienna, Austria | Division of Oncology, Department of Internal Medicine, Medical University of Graz, Graz, Austria | Department of Pathology, Donauspital, Vienna, Austria

Note: [] Corresponding author: Gernot Kriegshäuser, ViennaLab Diagnostics GmbH, Vienna, Austria. Tel.: +43 1 8120 15610; Fax: +43 1 8120 15619; E-mail: kriegshauser@viennalab.co.at

Abstract: We report the performance evaluation of a non-quantitative reverse-hybridization assay (KRAS-BRAF StripAssay) designed for the simultaneous detection of 10 mutations in codons 12 and 13 of the KRAS gene and BRAF mutation V600E. Dilution experiments using DNA from tumor cell lines or from formalin-fixed paraffin-embedded (FFPE) colorectal cancer (CRC) tissue were performed to assess assay sensitivity. Using 50 ng of total DNA (mutant and wild-type), the KRAS-BRAF StripAssay demonstrated a detection limit of 1% mutant sequence in a background of wild-type DNA. With respect to BRAF V600E, the KRAS-BRAF StripAssay was evaluated using 60 FFPE CRC samples previously analyzed by high resolution melting (HRM). Test strip hybridization identified 2/60 (3%) samples to carry the BRAF V600E mutation, and results were in agreement with those obtained by HRM analysis. This work demonstrates the KRAS-BRAF StripAssay to be a robust and sensitive method for the detection of common KRAS/BRAF mutations in genomic DNA isolated from FFPE tissue samples.

Keywords: KRAS, BRAF, mutation detection, reverse-hybridization, high-resolution melting

DOI: 10.3233/DMA-120960

Journal: Disease Markers, vol. 34, no. 3, pp. 171-177, 2013