High Resolution Melting (HRM)
High Resolution Melting (HRM) analysis is a relatively new, post-PCR analysis method used to identify variations in nucleic acid sequences. The method is based on detecting small differences in PCR melting (dissociation) curves. It is enabled by improved dsDNA-binding dyes used in conjunction with real-time PCR instrumentation that has precise temperature ramp control and advanced data capture capabilities. Data are analyzed and manipulated using software designed specifically for HRM analysis
Advantages of HRM
- Low reagent consumption, with little waste: HRM requires only PCR reaction volume (20 µL) 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 curve can be added to the end of the amplification run and analyzed immediately after completion
- Fast optimization: unlike DHPLC, thermal optimization is not required
- Low sample consumption: following HRM analysis, the PCR amplicon can be used directly in a Sanger sequencing reaction
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HRM analysis can also be used to detect methylation differences between genes; this is known as methylation-specific high resolution melting (MS-HRM) analysis.
Minimal temperature shift of DNA melting due to dye binding.
High Resolution Melt analysis can be used to scan for mutations in target genes for the identification of variant samples prior to sequencing analysis.
For information on Applied Biosystems solutions for HRM analysis - including instrumentation, software, and reagents.