HRM is an innovative technique that characterizes double-stranded PCR products based on their dissociation (melting) behavior as they transition from double-stranded DNA (dsDNA) to single-stranded DNA (ssDNA) with increasing temperature. Amplification of bisulfite-converted DNA is carried out using primers that are conversion-specific but not methylation-specific. The primers should therefore comprise several converted cytosines and should flank CpG sites (see figure "Principle of methylation analysis of the APC promoter by HRM"). This is to ensure amplification of bisulfite-converted DNA only and enable distinction between methylated and unmethylated CpG sites during HRM analysis.
The fluorescence of EvaGreen is measured continuously and is plotted against increasing temperature. EvaGreen is only measured as long as it is bound to dsDNA. This results in high fluorescence at the start of HRM analysis. As the increasing temperature gives rise to DNA melting, EvaGreen is released and the fluorescence reduces to a background level. GC-rich stretches of DNA are more stable and therefore melt more slowly compared with AT-rich regions and remain double stranded at higher temperatures for longer (see figure "Principle of HRM technology").
HRM analysis is an easy and cost-effective alternative to probe-based methylation assays. PCR products can be discriminated according to sequence, length, GC content, or strand complementarity — down to single base pair changes.
Convenient master mix format
The EpiTect HRM PCR Kit is provided in a convenient master mix format for increased ease-of-use. The optimized 2x EpiTect HRM PCR Master Mix ensures highly specific amplification. In addition, it enables flexible, rapid, and sensitive analysis of methylation status of CpG dinucleotides in bisulfite converted DNA via HRM. The master mix consists of optimized concentrations of HotStarTaq Plus DNA Polymerase, EpiTect HRM PCR Buffer, dNTPs, and EvaGreen dye.
HotStarTaq Plus DNA Polymerase
HotStarTaq Plus DNA Polymerase, a modified form of QIAGEN Taq DNA Polymerase, is supplied in an inactive state that has no polymerase activity at ambient temperature. This prevents extension of nonspecifically annealed primers and primer dimers formed at low temperatures during PCR setup and the initial PCR cycle.
Optimized EpiTect HRM PCR Buffer
EpiTect HRM PCR Buffer maintains specific amplification in every cycle of PCR. The balanced combination of KCl and (NH4)2SO4 in the buffer promotes specific primer–template annealing. Simultaneously, nonspecific annealing is reduced, maximizing yields of specific PCR product, which allows optimal HRM analysis. Specific amplification with high product yield is maintained without the need for time-consuming optimization of the Mg2+ concentration.