BRAFV600E Mutation Detection

The BRAF mutation testing assay utilizes PCR amplification and dye termination sequencing of exon 15 of the BRAF gene using specific PCR primers.

A histologic section of formalin-fixed paraffin embedded tissue is examined by a pathologist to identify an area of tissue with sufficient tumor for detection (≥40% tumor if possible). DNA is extracted from tumor areas on adjacent unstained slides and exon 15 of the BRAF gene is amplified in a PCR reaction. PCR products were purified using the Exo/SAP method. Sequencing reactions were performed using Big Dye v3.1 (Applied Biosystems) with M13 forward and reverse primers. The sequencing products are separated by capillary electrophoresis on an Applied Biosystems 3500XL Genetic Analyzer. Sequence traces were analyzed using Mutation Surveyor (Softgenetics).

Mutations are reported based on National Center for Biotechnology Information Reference Sequence: {"type":"entrez-nucleotide","attrs":{"text":"NM_004333.4","term_id":"187608632","term_text":"NM_004333.4"}}NM_004333.4.

The limit of detection for Sanger sequencing has been reported to be approximately 20% mutant allele.