Modified low cost SNP genotyping technique using cycle threshold (Ct) & melting temperature (Tm) values in allele specific real-time PCR

BACKGROUND & OBJECTIVES: Genotyping has now become one of the major diagnostic means for almost all diseases. Among the advanced techniques that are used to study single nucleotide polymorphisms (SNPs), only a few are applicable for routine disease diagnosis. Their applicability mainly depends on three factors: cost, time, and accuracy. The primary objective of this study was to propose allele-specific real-time PCR as a rapid, low cost and simple genotyping method for routine diagnostics. METHODS: Two SNPs, rs3014866 and rs2149356 were analysed using allele-specific real-time PCR. The polymerase chain reaction was carried out using RealQ PCR master mix containing SYBR Green DNA I dye followed by melt curve analysis. The results were validated by agarose gel electrophoresis and DNA sequencing. RESULTS: The allelic discrimination and zygosity of the two SNPs were assessed by combined cycle threshold (Ct) and melting temperature (T(m)) values. Variations in Ct and T(m) values among the two alleles were observed in both rs3014866 (Ct: C allele - 24±1, T allele - 27±1; T(m): C allele - 82.5±0.3, T allele - 86.3±0.2) and rs2149356 (Ct: C allele - 24±1, A allele - 26±1; T(m): C allele - 79.4±0.2, A allele - 80.4±0.3). Based on the variations, homozygous and heterozygous alleles were detected. Agarose gel electrophoresis and DNA sequencing also confirmed the allelic variation and zygosity observed in real-time PCR. INTERPRETATION & CONCLUSIONS: In diagnostic settings where a large number of samples are analysed daily, allele-specific real-time PCR assay may serve as a simple, low cost and efficient method of genotyping.