Principle of 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
To perform HRM, the temperature is increased from low to high. The fluorescence of third-generation intercalating dyes (e.g., EvaGreen) is measured continuously and is plotted against increasing temperature. Increased fluorescence is only measured as long as the dye is bound to dsDNA. At lower temperatures, fluorescence is high since all DNA is double-stranded. As the temperature increases, the DNA will start to disassociate into two single strands, resulting in DNA melting. At this point, the dye is released and the fluorescence will decrease. The melting temperature (T_m) of the DNA sample under analysis occurs at the point of the melt phase where the rate of change in fluorescence is greatest. When the DNA is completely melted, only some background fluorescence will be detected (see figure A typical HRM plot).

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
HRM is made possible not only by specialized instrumentation and software, but also by the introduction of third-generation fluorescent dsDNA dyes (see figure SYBR Green I versus EvaGreen dye). Third-generation intercalating dyes such as EvaGreen have been successfully used for HRM analysis on various real-time cyclers. These dyes have low levels of inhibition and can therefore be used at higher concentrations for greater saturation of the dsDNA sample. Greater dye saturation means that the fluorescent signals measured have higher fidelity, because there is less dynamic dye redistribution to nondenatured regions of the nucleic acid strand during melting, and because dyes do not favor products with a higher melting temperature profile (Wittwer et al. 2003). The combination of these characteristics provides greater melt sensitivity and higher resolution melt profiles, resulting in more distinct melting curves.

Using HRM to study base pair differences
HRM can be used to even detect single base pair differences (see figure HRM analysis of homozygous and heterozygous samples), as seen in complex applications such as SNP detection, mutation analysis, as well as microbial typing.

One of the most frequent applications of HRM is SNP genotyping. SNPs are single nucleotide variations in the genome with a frequency higher than 1% in a given population. These variations indicate how individuals respond to diseases, environmental factors, and drugs. SNPs are typically divided into four different classes (see table SNP classification), depending on the type of base exchange. A simple exchange of an A by a T will have only a very subtle influence on the T_m, but will still affect the melting behavior of the amplicon.

SNP classification

* Abundance in humans according to Venter et al. 2002.

SNP class	Base change	Typical Tm shift	Abundance (in humans)*
I	C/T and G/A	Large: >0.5°C	64%
II	C/A and G/T		20%
III	C/G		9%
IV	A/T	Very small: <0.2°C	7%

Using HRM for methylation analysis
HRM can be successfully used to detect changes in the methylation status of DNA.

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