Backgroundand aim: The V600E mutation is the most common BRAF gene mutation found in human cancers. However, discrimination between mutant and wild-type genes is crucial for the diagnosis of diseases such as breast or thyroid cancers, and to evaluate response to a specific treatment. For cancer patients, the tissue biopsy is a standard procedure used to pinpoint cancer’s mutations and degree of malignancy. Tissue biopsies may not always be easily obtained and often may involve the use of more invasive procedures. However, serum biomarkers as alternatives to tissue biopsy is a promising alternative to tissue biopsy. Cell-free DNA (cfDNA) is a free fragment released from various cells in plasma which can be used as a non-invasive biomarker in the diagnosis of cancer and genetic diseases. Due to the presence of the low levels of mutant versus wild-type, it is difficult to distinguish between the alleles. Using Allele-Specific PCR and Non-Extendable Primer Blocker Methods (NEPB) methods, this study was designed to discriminate the wild-type frommutant BRAFV600Eallels.
Method:DNA was extracted from human thyroid carcinoma (B-CPAP) and human breast cancer (MCF-7) cell lines, as mutant and wild-type controls. The allele-specific TaqMan real-time PCR assay was performed for amplifying a 100 bp target sequences in the BRAF gene. To prevent wild-type amplification and increasing the detection level of BRAF mutants, different concentrations of non-extendable primer blockers (NEPB) were added to the reaction mixture. The sensitivity and specificity of tests wereassessed by various concentrations of genomic DNA. As a template of cfDNA, DNA extracted from plasma samples collected from normal subjects using four distinct methods including phenol-chloroform, NaI, THP, and Roche commercial kit. The limit detection of the primers for the wild-type and the mutant genes were assessed by real-time PCR performed on serial dilutions of PCR amplicons.
Result: NEPB method was able to preven
