HRM is a closed-tube, post-PCR analysis method that has raised enormous scientific interest. 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 easily analyzed (see figure " Highly accurate genotyping"). HRM is easier and more cost-effective than probe-based genotyping analysis and, unlike conventional methods, it prevents carry-over contamination of PCR products.
The unique kit components ensure highly specific amplification (see table).
Novel EvaGreen dye for distinct melting curves
The Type-it HRM PCR Kit contains EvaGreen, a third-generation, saturating fluorescent dye which selectively binds to double-stranded DNA. In contrast to conventional SYBR® Green I, EvaGreen can be used at higher concentrations without PCR inhibition and shows equal binding affinity for GC-rich and AT-rich regions with no apparent sequence preference. This makes EvaGreen highly suitable for HRM analysis of all types of PCR products, enabling distinct melting curves due to visualization of lower fluorescent differences, thereby ensuring standardized results.
Optimized HRM PCR Master Mix
HRM PCR Master Mix consists of HotStarTaq Plus DNA Polymerase and an innovative HRM PCR buffer system, both of which enable highly specific amplification and distinct melting curves of even difficult genomic loci such as Class IV SNPs (see figure " Successful genotyping of an A/T Class IV SNP"). Specific amplification of the desired PCR product is ensured and generation of nonspecific products and formation of primer–dimers is minimized, if not eliminated (see figure " Successful typing of gene mutations").
The innovative buffer (included in the master mix) maintains specific amplification in every cycle of PCR by promoting a high ratio of specific-to-nonspecific primer binding during the annealing step in each PCR cycle. Owing to a uniquely balanced combination of KCl and (NH4)2SO4, the buffer provides stringent primer-annealing conditions over a wider range of annealing temperatures and Mg2+ concentrations than conventional PCR buffers. Optimization of PCR by varying the annealing temperature or the Mg2+ concentration is therefore often minimal or not required.
Suitable for difficult mutation loci
The Type-it HRM PCR Kit is a powerful system for easy detection of difficult mutation loci (see figure " Highly specific and successful amplification of difficult genomic loci"). Mutations or SNPs located in GC-rich regions or regions of high secondary structure are difficult to amplify using conventional HRM PCR solutions. The 2x HRM PCR Master Mix includes the innovative PCR additive, Q-Solution, at a defined concentration to help overcome these challenges. Q-Solution improves PCR by modifying the melting behavior of DNA, resulting in successful amplification even of difficult target sequences without the need for optimization.
Features of the Type-it HRM PCR Kit
|2x HRM PCR Master Mix ||Eliminates the need for optimization in the development of new assays |
Specially developed for HRM analysis of mutations and SNPs
Dedicated for use with all cyclers suitable for HRM analysis
Convenient reaction setup, minimizing pipetting errors
|HotStarTaq Plus DNA Polymerase ||Highly specific amplification |
Fast and easy room-temperature reaction setup
|Type-it HRM PCR Buffer ||Distinct melting curves |
Increased amplification specificity
|Q-Solution ||Improves amplification of difficult templates|
|EvaGreen Dye ||Novel, saturating dsDNA-binding dye|
Distinct melting curve analysis