Evaluation of SYTO13 as fluorescent dye for high resolution melting based single nucleotide polymorphism genotyping

Abstract

Objective To evaluate the utility of fluorescent dye SYTO13 for high-resolution melting (HRM) detection in single nucleotide polymorphism (SNP) genotyping and its clinical application. Methods This is a performance verification study. 36 genotype defined samples were divided into three groups: SNP rs3125734 C>T (class Ⅰ SNP) , rs255758 A>C (class ⅡSNP) and rs688C>T. These samples were used to evaluate SYTO13′s SNP genotyping capability of class Ⅰ SNP, class Ⅱ SNP, and two PCR products of different lengths (52 and 107 bp) covering the same SNP of rs688C>T. The commercial HRM dye of LCGreen Plus was used as the control. The genotyping capability is indicated by the Tm difference(ΔTm) between wild type and homozygous mutant genotypes. The Tm differences between wild genotype and homozygous mutant genotype were compared using the Independent Samples t test. Paired t test was used to evaluate genotyping capability of the two dyes. The clinical applicability is evaluated by synchronously performing PCR amplification and HRM analysis on thirty-five randomly selected DNA samples with known genotypes of the three SNPs. Results The SNPs of class Ⅰ and class Ⅱ can be genotyped directly and clearly with SYTO13 (ΔTmclassⅠ=0.36±0.05, tclassⅠ =14.827, PclassⅠ=0.000; ΔTm classⅡ=0.42±0.110, tclassⅡ=9.539, PclassⅡ=0.000). The classⅠSNP genotyping results was better using SYTO13 (ΔTmSYTO13=0.39±0.027), while the SNP genotyping for small amplicon did not discriminated clearly in this study. Long amplicons of class Ⅰ and Ⅱ SNPs can be identified directly except for several samples which can be genotyped accurately after having performed reexamination. Conclusion SYTO13 can apply for HRM analysis of genotyping classⅠ and Ⅱ SNPs with long amplicon and for clinical routine detection.(Chin J Lab Med, 2017, 40: 88-94) Key words: Polymorphism, single nucleotide; Genotyping techniques; Fluorescent dyes; High resolution melting