Abstract 411: Sensitive detection of BRAF V600E mutation by bridged nucleic acid (BNA)-mediate real-time PCR assay

Abstract

Abstract BRAF is a human gene encoding a serine/threonine protein kinase in the RAS-RAF-MEK-ERK signal pathway, which is important in regulating cellular responses to extracellular signals. Frequent mutations on BRAF gene have been detected in various types of human cancer, including malignant melanoma, non-small cell lung cancer, colorectal cancer, papillary carcinoma of the thyroid, ovarian cancer and hairy cell leukemia. Among all, a single point 1799T>A (V600E) transversion, the most common mutation on BRAF, results in increased kinase activity. It serves as a driver mutation in several types of cancer and confers increased sensitivity to tyrosine kinase inhibitors. Thus, detecting the BRAF V600E mutation may offer valuable information for cancer diagnosis, treatment selection, and prognosis. Here we report a rapid, highly sensitive method for the detection of BRAF V600 mutation using a bridged nucleic acid (BNA) clamp technology associated with quantitative PCR (qPCR). Due to the feature of enhanced affinity for hybridization template, BNA clamp is able to selectively block PCR reactions on the perfect match DNA sequence, while leaves the mismatch template unaffected. In this study several BNA clamps that bind to BRAF V600 wild-type (WT) template was designed, synthesized, and characterized. As compared to DNA clamp counterparts, these BNA clamps increased Tm values for the BRAF V600 WT by greater than 30°C. In addition, BNA clamps efficiently distinguished BRAF WT (perfect match) from V600E (mismatch) templates (deltaTm >12°C). In qPCR assays BNA clamps could suppress amplification of WT by >1,000-fold, while had little or no effects on the V600E mutant. Such BNA clamps allowed us to detect 1.0% or lower levels for the BRAF V600E mutant in a wild-type background. These results indicate that BNA clamp-based qPCR technology offers a new tool for rapid, sensitive detection of BRAF V600E mutation. The technology may be adapted for detection of mutations of a variety of biomarker genes. Citation Format: Xiaoyun Liu, Leticia Loredo, Houquan Dai, Aaron Castro, Yuewei Zhao, Sung Kun Kim, Austin Dinkel, Miguel Castro. Sensitive detection of BRAF V600E mutation by bridged nucleic acid (BNA)-mediate real-time PCR assay. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 411.