BRAF V600E Mutation Detection Using High Resolution Probe Melting Analysis

Activation of oncogenic proteins is an important mechanism in carcinogenesis. The BRAF gene, located on chromosome 7q34, encodes a serine-threonine kinase that acts downstream of RAS in the RAS/RAF/MEK/ERK signaling pathway involved in regulating cell proliferation and survival. On activation of RAS, the BRAF kinase is activated and sequentially phosphorylates and activates MEK and ERK. A mutation in BRAF leads to constitutive hyperactivation of this pathway through evasion of the inhibitory feedback loop resulting in increased ERK signaling output which drives proliferative and anti-apoptotic signaling (Pratilas et al. 2009). Mutations in BRAF have been reported to occur at high frequency (66%) in melanoma with lower frequencies in colon and other tumours (Davies et al. 2002); BRAF is thus considered to be an important therapeutic target in melanoma (Bollag et al. 2010; Flaherty et al. 2010; Paraiso et al. 2011). Although over 30 single site missense mutations have been identified, 90% occur at nucleotide 1799 resulting in a T-A transition and an amino acid substitution at residue 600 (V600E) in the activation segment (Wan et al. 2004).