Principle of HRM Technology
Simplify your genetic analysis with innovative HRM technology
HRM technology description
HRM analysis is based on the dissociation behavior of dsDNA due to increasing temperature. Melting of dsDNA depends on its GC content and overall distribution of bases. AT-rich regions melt faster (see figure Principle of HRM technology).
In HRM analysis, a PCR product is generated through amplification and is then subjected to a gradual temperature increase. This is done in the presence of a dye that fluoresces when bound to double-stranded DNA. This method clearly differs from standard melt curve analysis (see figure Differences between classical melt curve analysis and HRM analysis). Novel HRM technology is enabled by the recent availability of improved double-stranded DNA (dsDNA) binding dyes along with dedicated HRM instrumentation and analysis software.
Typical HRM experiment
DNA strands of a PCR product are bound together by hydrogen bonds and additional interactions such as base stacking forces. The strength of these interactions depends on parameters such as the length of the amplicon, the overall base composition, and the local GC content within the PCR amplicon. All of these parameters will affect the melting behavior of a defined PCR product. Each PCR product will deliver a characteristic melting profile (see figure Principle of HRM technology).
HRM even enables the detection of very subtle differences between two DNA sequences. It thereby allows the accurate detection of variations in the DNA sequence down to the single nucleotide level.
Role of the intercalating dye
Using HRM to study base pair differences
|SNP class||Base change||Typical Tm shift||Abudance (in humans)*|
|I||C/T and G/A||Large: >0.5°C||64%|
|II||C/A and G/T||20%|
|IV||A/P||Very small: <0.2°C||7%|
Using HRM for methylation analysis
Amplification of bisulfite-converted DNA is carried out using primers that are conversion-specific, but not methylation-specific. The primers should therefore comprise several converted cytosines and should flank CpG islands (see figure Principle of methylation analysis of the APC promoter by HRM). This is to ensure amplification of bisulfite-converted DNA only and enable distinction between methylated and unmethylated CpG sites during HRM analysis. GC-rich stretches of DNA are more stable and melt more slowly compared to AT-rich regions and remain double stranded at higher temperatures for longer (see figure Principle of HRM technology).
Versatile HRM technology
HRM is a universal technology applicable in a multitude of research fields, including:
- Typing of disease and cancer loci
- Biomarker discovery
- Typing of transgenic plants and animals
- Pathogen detection and genotyping
- Methylation studies