High Resolution Melting, HRM, is a post-PCR, closed-tube technique that can be used to analyze genetic variations. HRM allows you to rapidly perform single nucleotide polymorphism (SNP) or point mutational analysis, zygosity testing and even epigenetic analysis. With traditional technologies, following DNA amplification the sample is loaded on a matrix (or gel) for electrophoresis, dHPLC, Mass Spec or array analysis, to separate and detect any changes. With HRM, following target amplification, a melting step under high-resolution conditions enables SNP detection or discovery in the same run, saving time and money.
How does HRM work? HRM analysis relies on the presence of a saturating, double-stranded DNA binding dye during the thermal denaturation of a double-stranded DNA amplicon and an extremely well controlled thermal denaturation profile. The dye intercalates into the dsDNA helix structure in a base-pair dependent manner, and fluoresces strongly only when intercalated. After amplification in the presence of the dye, the DNA products are briefly melted and then allowed to re-anneal. This results in the production of all possible homo- and hetero-duplexed amplicons. The gradual re-melt of these amplicons reveals the differences in the base-pairing of amplicons by changes in the shape of the melt profiles.
What do you need set up a HRM experiment? HRM requires a saturating, non-inhibitory, dsDNA binding dye, a high performance PCR instrument, and data analysis software. Amplicon melting temperature differences between a wild type and mutant are in the range of 0.2 to 0.5 degrees, so an instrument used for HRM should have a very low delta temperature across the entire block. Critical components of experimental optimization include primer design, sample preparation, reaction optimization, and data analysis.
A major advantage of HRM is the ability to scan for mutations without the need for sequencing. HRM allows any amplicon to be screened for unknown variants; allele-specific primers or probes are not required. HRM can also be used as an alternative to Next Generation Sequencing for methylation analysis. In the presence of controls and standards the percent methylation of experimental samples can be determined based on melting curve analysis and melting temperature changes in the sequence of interest.
For high throughput analysis, HRM is a cost effective genotyping technology, delivering fast and robust results.
Watch Video:
In this Bench Tip Video, Dr. Mike Okimoto, Chief Content Officer Biocompare, discusses High Resolution Melting.
Related Products from: Roche Applied Science