This qPCR mix delivers maximum specificity to your high resolution melt (HRM) curve analyses. Accurately detect genetic mutations, quickly identify genotypes based on SNPs, or calculate percent methylation of a target region with HRM analysis.
Clara™ HRM Mix comprises this unique, ultra-pure, PCRBIO Taq DNA Polymerase in a blend containing dNTPs and MgCl2. Powered by a third-generation, DNA-intercalating, SyGreen 2 dye for greatly reduced PCR inhibition, this new generation high-resolution melting qPCR mastermix offers superior performance for accurate SNP discrimination and quantification of methylation differences.
- Accurate distinction of SNP classes I-IV
- Quantify methylation of target sequences
- Super-sensitive product melt curves for distinct allele profiles
- Compatible with all HRM-suitable real-time instruments
- Powered by PCRBIO Taq DNA polymerase
High Resolution Melt (HRM) curve analysis is a powerful technique used for the analysis of mutations, polymorphisms and epigenetic differences in double stranded DNA samples. This method relies on analysis of the post amplification denaturation of qPCR targets in combination with third generation DNA intercalating dyes with reduced polymerase inhibition (e.g. SyGreen 2, EvaGreen®). HRM analysis exploits the differences in melt curve shapes and DNA melting temperature to discriminate sequence variations between samples.
Usually, in a melt curve analysis run after a standard dye-based qPCR, fluorescence data is acquired every 0.2-0.5 °C, whereas in HRM analysis data is acquired at a much higher density, every 0.1 °C, resulting in far greater resolution of the resulting melt curve. Thus, even small difference (down to single point mutations, SNPs, and base-pair methylation), in target sequences register as a shifted melt curves. HRM thus offers a cheaper effective alternative to probe-based detection for SNP detection, allelic discrimination or target variant detection.
HRM compatible real-time instruments
Running HRM experiments requires compatible instrumentation. Not all real-time PCR instruments are able to achieve the necessary temperature increment to conduct HRM profiling. We recommend using one the listed qPCR thermocyclers from the following manufacturers:
- Applied Biosystems: 7900, 7900HT, 7900HT FAST, StepOne™, StepOne™ Plus, 7500, 7500 FAST, Viia7™
- Bio-Rad®: CFX96™, CFX384™
- Eppendorf: Mastercycler® ep realplex, Mastercycler® realplex 2S
- Qiagen/Corbett: 6000, Q
- Roche Applied Science: Lightcycler®480, Lightcycler®Nano
Other instruments that are not listed are also suitable for HRM analysis using Clara™ HRM Mix. Use our qPCR Selection Tool, check your instrument's product manual, or contact us for help in choosing an appropriate instrument.
Detectable sequence modifications
Clara™ HRM Mix can distinguish all types of SNPs. These include:
- Class I: C to T and G to A
- Class II: C to A and G to T
- Class III: C to G
- Class IV: A to T
Additionally, this mix can also be used to quantify CpG methylation in a target sequence. In this case, potentially methylated DNA is subjected to bisulfite treatment prior to running HRM qPCR with primers targeting CpG containing regions. During bisulfite treatment, all unmethylated cytosine residues are deaminated and thus converted to uracil residues, while all methylated cytosine residues remain unaltered. HRM analysis on the bisulfite treated versus a corresponding, untreated, control sample allows the estimation of the target’s cytosine methylation percentage, because methylated sequences will have a higher CG content compared to unmethylated targets following bisulfite treatment.
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