Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
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SHR Neuro Krebs Kardio Lipid Stoffw Microb
Pichler, M; Balic, M; Stadelmeyer, E; Ausch, C; Wild, M; Guelly, C; Bauernhofer, T; Samonigg, H; Hoefler, G; Dandachi, N.
Evaluation of high-resolution melting analysis as a diagnostic tool to detect the BRAF V600E mutation in colorectal tumors.
J Mol Diagn. 2009; 11(2):140-147
Doi: 10.2353/jmoldx.2009.080100
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- Führende Autor*innen der Med Uni Graz
- Balic Marija
- Dandachi Nadia
- Pichler Martin
- Co-Autor*innen der Med Uni Graz
- Bauernhofer Thomas
- Gülly Christian
- Höfler Gerald
- Samonigg Hellmut
- Stadelmeyer Elke
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- Abstract:
- BRAF V600E is the predominantly occurring mutation of the cytoplasmic kinase BRAF, and, in colorectal cancer, its determination provides a diagnostic exclusion criterion for hereditary nonpolyposis colorectal cancer. The aim of our study was to develop a sensitive BRAF V600E high resolution melting (HRM) assay. We first established and optimized the BRAF HRM assay using a cell line dilution model, enabling us to detect 1% mutant DNA in a background of wild-type DNA. In a comparison, DNA sequencing and real-time allele-specific PCR in the cell line dilution model HRM assay proved to be more sensitive than DNA sequencing and denaturing high performance liquid chromatography, retaining the same sensitivity as real-time allele-specific PCR. In a learning set of 13 patients with known BRAF V600 status, the mutation was detected with high concordance by all four methods. Finally, we validated the HRM assay on 60 formalin-fixed, paraffin-embedded colorectal cancer samples. Although all mutated samples were correctly identified by HRM, the detection limit of the HRM assay decreased when using low-quality DNA derived from formalin-fixed, paraffin-embedded samples. In conclusion, HRM analysis is a powerful diagnostic tool for detection of BRAF V600E mutation with a high sensitivity and high-throughput capability. Despite the expected decrease in sensitivity, HRM can reliably be applied in archival formalin-fixed, paraffin-embedded samples tissues.
- Find related publications in this database (using NLM MeSH Indexing)
- Adult -
- Aged -
- Aged, 80 and over -
- Cell Line, Tumor -
- Colorectal Neoplasms - diagnosis Colorectal Neoplasms - genetics
- DNA Mutational Analysis - methods
- Formaldehyde - chemistry
- Humans -
- Middle Aged -
- Molecular Diagnostic Techniques - methods
- Proto-Oncogene Proteins B-raf - genetics
- Reproducibility of Results -
- Sensitivity and Specificity -
- Tissue Fixation -
- Transition Temperature -