Enhanced Specificity of BRAF V600E Genotyping Using Wild-Type Blocker Coupled with Internal Competitive Reference in a Single Tube.

BACKGROUND: Mutations in the BRAF gene have been strongly associated with failure in cancer treatment using epidermal growth factor receptor (EGFR) antibodies. To better diagnose and assess the prognosis of cancer patients, mutation screening of the BRAFV600E gene should be performed prior to clinical anti-tumor drug therapy to avoid ineffective treatment.

METHODS: In our previous study, we developed a real-time wild-type blocking PCR (WTB-PCR), which can amplify the mutant allele at high efficiency while simultaneously inhibiting the amplification of wild-type alleles. In order to reduce base mismatch due to the high number of cycles, as well as to monitor the total quantity of DNA added to the reaction system, an internal reference gene was co-amplified together with the target gene on the basis of WTB-PCR.

RESULTS: Our results showed that when 50 - 200 ng of the DNA templates was used, this current built method (realtime quantitative clamp-based PCR technology using wild-type blocker coupled with internal competitive reference to enhance amplification specificities, named wirePCR) completely blocked the amplification of the wild-type BRAFV600E gene with detection of the mutated allele at a mutant/wild-type ratio of 1:10,000, which was in line with the sensitivity requirement for the detection of trace amounts of the mutant gene. In the colorectal biopsies from 50 patients with suspected colorectal cancer, eight patients (16%) with BRAFV600E mutations were detected using wirePCR. The allele percentage of mutations can be obtained directly from the ΔCq between the targeted and reference genes, we demonstrated that among the V600E-positive patient samples, the percentage of BRAF DNA with the V600E mutation ranged from 24.99% to 54.31%.

CONCLUSIONS: WirePCR is a rapid, simple, and low-cost quantitative analytical technique for the detection of trace amounts of mutant BRAFV600E genes in clinical samples.