Background: Molecular detection of 1799 T>A point mutation in BRAF gene is prognostic and diagnostic for papillary thyroid carcinoma (PTC 70%). Furthermore, BRAF inhibitor molecules like vemurafenib are proving highly effective to treat patients with BRAF mutated tumors, in melanoma; in the next future these approach could be suitable for PTC treatments. This can prove a challenge and underlines the importance of utilizing a detection method that is easy, sensitive, reproducible and cost-effective.
Materials and Methods: We developed a cost-effective and sensitivity method based on Locked Nucleic Acid (LNA) oligomers for selective detection of BRAF 1799T>A V600E. LNA have been used to enhance mutant allele of minor background variants in a large excess of wild type allele derived from DNA extracted by of 14 thyroid biopsy; 14 of them obtained from cytological slides and 5 from fresh Fine Needle Aspirate (FNA) of patients with papillary thyroid carcinoma (PTC). In addition, we compared the results with those obtained by direct sequencing.
Results: The assay sensitivity of the method was 0.1% of mutant alleles, assessed by serial dilution of DNA from ARO cell line (carrying heterozygous V600E), mixed to wild type DNA obtained from Healthy donors. Optimized concentration of the primer LNA clamping-wild type (wt) DNA is 12 µM per reaction. Direct sequencing was able to detect mutation in only 26.7% (5/19) while, LNA-based PCR assay 57.9% (11/19) of patients, carrying mutation at codon V600. The estimated reagents costs is about € 20,00 per sample, including controls and pre-analytical steps. This assay could be performed in a simple thermalcycler and results visualized by agarose ethidium bromide stained gels.
To cite this article
A cost-effectiveness method for the enrichments of minority mutant DNA in a large excess of wild type DNA. Detection of B-RAF 1799T>A (V600E) mutations in the thyroid fine needle aspirate
WCRJ 2014; 1 (4): e399
Published online: 21 Dec 2014
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