Sensitive detection of BRAF V600E mutation by Amplification Refractory Mutation System (ARMS)-PCR

To detect BRAF V600E mutation in formalin-fixed, paraffin-embedded (FFPE) tissue, we developed a method using Amplification Refractory Mutation System (ARMS)-PCR. This method was designed to amplify three products in a single reaction tube: a 200 bp common product serving as an amplification control, a 144 bp BRAF V600E specific product, and a 97 bp wild-type (wt) specific product. The sensitivity of this method was determined to be as low as 0.5% for the BRAF V600E allele in a wild-type background. This method was successfully validated in 72 thyroid tumors. It detected V600E mutation in 22 out of 33 (67%) of the conventional papillary thyroid carcinoma (PTC), 8 out of 12 (75%) of the tall-cell variant of PTC, whereas none of the 10 follicular variant of PTC showed BRAF V600E mutation. In addition, none of the 14 follicular adenomas and 3 follicular carcinomas had BRAF V600E mutation. As a comparison method, direct dideoxy sequencing found only 27 out of 30 (90%) mutations detected by ARMS-PCR method, suggesting that this ARMS-PCR method has higher sensitivity.Our ARMS-PCR method provides a new tool for rapid detection of BRAF V600E mutation. Our results indicate that ARMS-PCR is more sensitive than automated dideoxy sequencing in detecting low BRAF V600E allele burdens in FFPE tumor specimen. The strategy of this ARMS-PCR design may be adapted for early detection of point mutations of a variety of biomarker genes.BRAF gene encodes a serine/threonine protein kinase that functions downstream of RAS in the RAS-RAF-MEK-ERK signaling pathway, also known as mitogen activated protein kinase (MAPK) pathway, which is important in regulating cellular responses to extracellular signals including epidermal growth factor (EGF) [1]. Upon activation by receptor binding, RAS proteins recruit RAF to the cellular membrane where RAF is phosphorylated [2]. Activated RAF proteins further phosphorylate MEK1 and MEK2, which then phosphorylate ERK1 and ERK2. Phosphorylated ERK proteins r