Optimization of BRAF V600 Assay as A 2-Step Real-time PCR Protocol

Abstract

The QClamp® BRAF Codon Specific Mutation Detection Kit is a real-time PCR assay for the detection of somatic mutations in codon 600 Valine at exon 15 in the BRAF gene which encodes the serine/threonine protein kinase, using purified DNA. The V600E mutation is the most common BRAF gene mutation found in human cancers. This mutation leads to production of a BRAF protein that is abnormally active, which disrupts regulation of cell growth and division. Mutations in this gene have been found in cancers, including non-Hodgkin lymphoma, colorectal cancer, malignant melanoma, papillary thyroid carcinoma, non-small-cell lung carcinoma, gastric cancer, and even prostate cancer. Currently, the established qPCR protocol for the QClamp® BRAF Mutation Detection Assay is comprised of a 4-step procedure: Denaturation, XNA Annealing, Primer Annealing and Extension. The purpose of this experiment was to test the feasibility of optimizing this assay to a more efficient and faster 2-step Real-time PCR which has just the Denaturation and the Primer Annealing/Extension steps. Optimization was attempted on both the ABI-QS5 and LC480 thermocycling instruments using parallel testing. The newly established 2-step thermocycling parameters were successfully tested and validated on the ABI-QS5 instrument. For the LC480, however, the experiment was not successful. This result might be due to the different platforms and technologies of the two instruments. Further research is needed to develop the mutational status scoring and acceptance criteria for clinical samples on the ABI-QS5, and to complete the development of the 2-step qPCR protocol individually on the LC480; and also to study the effects of factors such as temperature, ramp rates, PCR enzymes/master mix, primer/probes and XNA concentrations for both ABI-QS5, and the LC480