EpiTect HRM PCR Kit

Novel fluorescent dye EvaGreen

EvaGreen is a dsDNA-binding dye. The spectral properties of EvaGreen are similar to those of SYBR^® Green I. The absorbance maximum is 500 nm, and the emission maximum is 530 nm. In contrast to SYBR^® Green I, EvaGreen can be used at higher, saturating concentrations without PCR inhibition and shows equal binding affinity for GC-rich and AT-rich regions with no apparent sequence preference. This makes EvaGreen an ideal dye for HRM analyses of all types of PCR product.

Highly sensitive detection of changes in CpG methylation status

For CpG methylation analysis by HRM, the DNA has to undergo complete bisulfite conversion before the melting behavior of DNA can be measured by HRM analysis. Unknown samples can be compared with a standard control, based on their melting characteristics. The sensitivity ensured by the EpiTect HRM PCR Kit means that even low amounts of methylated DNA can be detected (see figure Highly sensitive results).

Consistent performance on various real-time instruments

The EpiTect HRM PCR Kit delivers highly accurate methylation results with real-time instruments suitable for HRM (see figure "Methylation analysis on various real-time PCR instruments"). The kit includes an optimized protocol that has been tested on the Rotor-Gene Q, Rotor-Gene 6000, and LightCycler 480. The combination of advanced HRM technology on the Rotor-Gene Q with the benefits of the EpiTect HRM PCR Kit delivers exceptional HRM analysis.

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.

The target sequence is PCR amplified to a high copy number in the presence of the dsDNA binding fluorescent dye, EvaGreen. The dye does not interact with single-stranded DNA (ssDNA), but actively binds to dsDNA and fluoresces brightly when bound. Following the amplification, the temperature is increased, and the fluorescence of EvaGreen is measured continuously and plotted against the temperature.

The EpiTect HRM PCR Kit provides a highly reliable and cost-effective method for methylation analysis using HRM technology and can be successfully used for methylation studies in epigenetic research. The optimized protocol means that information on the methylation status of CpG sites can be derived quickly and efficiently. This simplifies methylation assay development.