High Resolution Melt (HRM) analysis is a powerful tool for the detection of mutations and polymorphisms in dsDNA samples, including GpG methylation differences. At increasing temperature, in the presence of a fluorescent dye, tiny sequence variation between samples results in different DNA strand dissociation behaviour. HRM analysis exploits these differences using the melt curve shapes to distinguish between these samples.
Xpert Fast HRM (2X mastermix) contains all components, except specific primers and template, for HRM analysis in qPCR. The mix consists of the combination of a highly efficient enzyme with an optimized buffer system, which allows for efficient amplification of GC-rich and AT-rich templates under both normal and fast qPCR conditions. The intercalating dye used in this mastermix causes no inhibition of the PCR reaction thus allowing for extremely high sensitivity and specificity, as well as preventing the formation of unwanted primer-dimers and non-specific products.
Different SNPs show different Tm melt curve shifts. Class I SNPs (C/T and G/A) demonstrate typically large shifts (>0.5ºC), whereas Class IV SNP (A/T) typically have very small shifts (<0.2ºC). The high sensitivity and specificity of Xpert Fast HRM allows to distinguish all possible SNPs.
High Resolution Melt (HRM) Analysis
CpG methylation analysis (by comparing bisulfite-treated and control DNA)