High-resolution melting (HRM) is a closed-tube, post-PCR analysis method that has raised enormous scientific interest and has utility in diverse applications such as genotyping, epigenetics, and pathogen typing. HRM 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. PCR products can be discriminated according to sequence, length, GC content, or strand complementarity, down to single base pair changes. Previously unknown and even complex sequence variations, as seen in challenging genotyping applications, can be readily detected and characterized in an easy and straightforward manner (see figure Highly accurate genotyping by HRM analysis
). HRM is easier and more cost-effective than probe-based genotyping analysis and unlike conventional methods, it prevents carry-over post-PCR contamination.
HRM can be easily performed using the Rotor-Gene Q for applications such as:
- SNP genotyping
- Mutation detection
- Pathogen typing
- Methylation analysis
Predesigned assays for effortless evaluation of HRM technology
The Rotor-Gene Type-it HRM Discovery Kit demonstrates the detection of deletions or SNPs via high-resolution melting analysis. HRM technology enables rapid characterization of DNA samples based on their melting behavior following PCR amplification. The Rotor-Gene Type-it HRM Discovery Kit has been developed to exhibit the superior thermal and optical capabilities of the Rotor-Gene instrument in conjunction with the exceptional performance of the HRM PCR Master Mix. In addition to a master mix and template DNA, the kit provides 2 preoptimized HRM assays — a class IV A/T SNP (see figure Class IV SNP assay
) and a deletion assay (see figure Deletion assay
). Additionally, manual pipetting steps can be avoided by using the QIAgility, a compact bench-top instrument that provides rapid, high-precision PCR setup.
Novel EvaGreen dye for distinct melting curves
EvaGreen, a third-generation, saturating fluorescent dye that selectively binds to double-stranded DNA is included in the 2x HRM PCR Master Mix. In contrast to conventional SYBR®
Green I, EvaGreen can be used at higher concentrations without PCR inhibition and shows equal binding affinity for GC- and AT-rich regions, with no apparent sequence preference. This makes EvaGreen highly suited to HRM analysis of all types of PCR products, enabling distinct melting curves due to visualization of lower fluorescent differences, thereby ensuring standardized results.
Preoptimized HRM PCR master mix
The 2x HRM PCR Master Mix consists of HotStarTaq Plus DNA Polymerase and an innovative HRM PCR buffer system. The stringent hot start provided by chemically modified HotStarTaq Plus
DNA Polymerase allows reaction setup at room temperature. The unique composition of the HRM PCR Buffer ensures extremely stringent and targeted primer annealing, leading to highly specific amplification.