Let’s Get Melting

What is HRM Analysis?

It is now more than a decade since the introduction of melting analysis to characterize PCR products. Melting analysis following SYBR green-based real-time PCR has become a mainstay in research laboratories worldwide for applications such as gene expression because of it’s ease of design and cost-effectiveness (i.e. no need for expensive labeled probes).

Melting analysis detects variations in amplicons as small differences in dissociation curves of dsDNA amplicons post-PCR. This allows the detection of off-target PCR amplification without the need for agarose gels. Melting analysis also offers the advantage that it can discriminate between amplicons with very similar sequences, whereas agarose gels can only resolve PCR products according to size.

HRM analysis is an advancement on melting analysis, facilitated by the use of improved dsDNA-binding dyes in real-time PCR cyclers with precise temperature ramp control and advanced data acquisition capabilities. Newer PCR instruments include dedicated software for the analysis and manipulation of HRM data. Developments in real-time PCR dyes, PCR instrument heating precision and new software algorithms in recent years have led to HRM analysis becoming widely-established as a rapid, cost-effective and far-ranging means for rapid sequence analysis.

What Can We Use HRM Analysis For?

Originally described for single nucleotide polymorphism (SNP) variant analysis (and still the leading application), HRM analysis now boasts a much broader range of applications including human leukocyte (HLA) comparisons (e.g. HLA typing and HLA matching), microsatellite genotyping and methylation status of DNA sequences.

Within SNP analysis, HRM analysis has several applications – scanning regions for heterozygotes (and possibly homozygous variants) or for genotyping a known SNP. Newer developments such as unlabeled probes and snapback elements on PCR primers allow simultaneous genotyping of a desired SNP along with scanning of the amplicon for any other sequence variation(s).

HRM analysis is also useful in the rapid detection of known microbial drug-resistance genes in patient samples (e.g sputum, blood). To set up such an assay, a library of reference melt curves is prepared, containing all known drug-resistance genes/mutations associated with the microbe of interest. Melt curves obtained from patient samples are then compared to the reference library. Library preparation is typically carried out by cloning or mutating the genes of interest in a plasmid.

New methods and applications means that the exponential trend of HRM analysis publications is unlikely to plateau any time soon… so get melting!

Suggested Reading

1. Montgomery JL, Sanford LN, Wittwer CT. High-resolution DNA melting analysis in clinical research and diagnostics. Expert Rev. Mol. Diagn. 2010;10:219-40.
2. Erali M, Wittwer CT. High resolution melting analysis for gene scanning. Methods 2010;50:250-61.
3. Anything else by CT Wittwer
4. Vossen RH, Aten E, Roos A, den Dunnen JT. High-resolution melting analysis (HRMA): more than just sequence variant screening. Hum. Mutat. 2009;30:860-6.
   Highly recommended review describing a number of applications of HRMA

Originally published in 2010. Updated and republished in June 2017.