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High Resolution Melting (HRM) analysis is a post-PCR analysis method used to identify genetic variation in nucleic acid sequences.
The MeltDoctor™ HRM Master Mix contains all components (excluding template and primers) formulated for superior HRM performance across a wide range of genomic targets. Unlike some competitive mixes, the MeltDoctor™ HRM Master Mix does not require additional mixing prior to use, and was developed and optimized solely for HRM applications.
- Low background fluorescence
- High brightness in the presence of double-stranded DNA
- Minimal temperature shift of DNA melting due to dye binding
- Thermal stability to tolerate PCR cycling conditions
- No inhibition of polymerase activity, resulting in high PCR efficiency
- A dNTP blend including dUTP, which minimizes carryover contamination by allowing amplicon degradation by uracil DNA glycosylase (UDG) in subsequent PCR reactions
Perform Mutation Scanning
HRM analysis can be used to scan for mutations in target genes for the identification of variant samples prior to sequencing analysis (Figure 1). As a mutation scanning technique, HRM offers significant advantages over conventional methods such as Denaturing High- Performance Liquid Chromatography (DHPLC) and Denaturing Gradient Gel Electrophoresis (DGGE). Specifically, the advantages of HRM for mutation scanning include:
- Low reagent consumption, with little waste: HRM requires only a 20 µL PCR reaction for analysis of each sample, eliminating the need for HPLC solvents or DGGE gels
- Simple, fast workflow: no additional instrumentation is required after PCR amplification. A high-resolution melt step can be simply added to the end of the PCR profile for immediate analysis
- Fast optimization: unlike DHPLC, thermal optimization is not required
- Low sample consumption: following HRM analysis, the PCR product can be used directly in a Sanger sequencing reaction
- Magnesium salts and other buffer components, precisely formulated to obtain optimal HRM results. - AmpliTaq Gold® 360 DNA Polymerase, a highly purified DNA polymerase that provides hot-start performance, minimizing non-specific product formation and enabling reactions to be set up at room temperature
- MeltDoctor™ HRM Dye, a stabilized form of the fluorescent SYTO® 9 double-stranded nucleic acid stain developed by Molecular Probes.