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 detect ed. 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.