Modifying Tetra-Arms-PCR using melting curve analysis with sybr green (tapma) for genotyping SNP Rs11614913 in MIR - 196A2

Abstract

Single nucleotide polymorphism (SNP) is widely used as DNA markers for population genetic and mapping gene for complex disease. Despite many recent advances in high-throughput SNP genotyping, rapid, simple and inexpensive methods are still needed. In this study, we introduce a single-tube PCR method that combines Tetra-ARMS-PCR, melting curve analysis and SYBR Green dye to detect the SNP rs11614913 in mir-196a2. This assay, called TAPMA, is based on the melting point Tm and the melting curve patterns of the allele-specific fragments and non-allele-specific fragments to identify the genotypes of different samples. The primers were designed by a Web-based program and the TAPMA was optimized using blood samples from breast cancer patients to genotype the SNP of interest. Notably, the three control samples for three genotypes of SNP rs11614913 have been identified based on melting curve analysis and DNA sequencing. These samples will be used for further genotyping to analyse the association between this SNP and the breast cancer symptom in Vietnamese. Despite some disadvantages and limits during the optimizing step, the method initially showed satisfactory results in genotyping SNP rs11614913, but further optimization is still required to complete the method. We believe that TAPMA has a potential to become a useful tool for further research in SNP genotyping. Keywords: SNP Tetra-ARMS-PCR SYBR green Melting curve analysis TAPMA