P1.03 SNPS Detection in the Braf Gene
2012
Abstract Personalized medicine focused at individual patient's treatment by matching between the genetic information hidden at the DNA and medication. Single nucleotide mutations (SNPs) play a major role in genetic variability and the ability to react to medications.BRAF gene is involved in solid tumors like malignant melanoma and thyroid. BRAF is a serine/threonine protein kinase, activates the MAPK/ERK signaling pathway.The development of specific BRAF inhibitors that work only on the SNP V600E1 (T > A at position 1799) such as vemurafenib (Zelboraf) gave push to personalized medicine as 50% of metastatic melanoma patients carry this mutation. Those who carry the mutation demonstrate better response to vemurafenib than others. By comparison between 2 methods to detect SNPs in the BRAF gene, we show here that INFINITI™ Analyzer is the most accurate and reliable method. Methods BRAF mutation analysis by Length Analysis. We used allele-specific DNA with two specific forward primers for the mutant and the wild-type alleles and one common reverse primer. Mutated allele gives fragment of 160 bp and the wild type allele gives fragment of 157 bp. PCR was carried out and products were separated on sequencer and analyzed using peak scanner. BRAF mutation detected by AutoGenomics INFINITI-BRAF Assay. Following PCR amplification a removal of the non-incorporated dNTPs was performed.The products were loaded on BioFilmChip microarrays together with analyte specific primer extension fluorescently label. Results and Discussion We tested 101 samples and compare the results from the AutoGenomics INFINITI Assay with those of the DNA Fragment Analysis. The result reveals that the INFINITI BRAF Assay is sensitive and most easy to work with. That assay is almost fully automated and need shorter time for processing and lower concentration of DNA. The last issue is more important in cases of thyroid cancer were samples are taken by fine needle aspiration. We achieved 97% of concordance between both methods. All 3 discrepant cases were of V600E1 mutation detection that were found only by the INFINITI-BRAF. The ability of the INFINITI to detect mutation is 1 mutant cell I population of 100 cells. Both methods are suitable for SNP detection.
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