Since its inception in 2003 from a collaboration between the University of Utah and Idaho Technology, high-resolution melt analysis (HRMA) has been identified as a powerful technique used to generate DNA melt curve profiles for genotyping, mutation detection, and sequence comparison. The method is specific and sensitive enough to characterize nucleic acid sequence changes for both discovery of unknown genetic variation and routine detection of known variants.

Carl Wittwer, professor of pathology and adjunct professor of biomedical engineering at the University of Utah, who worked to develop the method calls HRM an accessible technology for screening multiple samples quickly. HRM was developed after observing changes in fluorescence during real-time PCR cycling, where Wittwer and colleagues noticed rapid drops in fluorescence when double-stranded DNA (dsDNA) melts. These observations led to the creation of HRM and the ability to obtain better precision in measuring real time melting and fluorescence than earlier melt analysis. Given its ease of use and high-quality data, HRMA has come to replace traditional analyses requiring electrophoresis or other less consistent techniques in extracting data from PCR products.

Benefiting from high-resolution melt analysis

High-resolution melting methods begin with the heating of dsDNA, where it unwinds into its single-stranded counterparts. The acquisition of data from this dissociation of dsDNA is used in HRM analysis. To start, a PCR amplicon to an area of interest is generated in the presence of a dye that only emits light when bound to dsDNA. As the temperature is increased and the dsDNA separates, the fluorescence decreases, creating a melting curve in real time that can be compared to a standard sequence to identify subtle genetic variations. “In contrast to classical melt curve analysis, HRM provides significantly more information, down to single nucleotide differences within less than an hour and at much lower costs than alternative methods for the identification of genetic variants. It is easy to use and flexible enough to be used in various applications,” describes Andreas Missel, Ph.D., director of research & development at QIAGEN.

Precision Melt Analysis is Bio-Rad’s implementation of HRM and expands the utility of qPCR systems by allowing variant study applications for minimal additional cost. “By utilizing relative melting differences, scientists can compare small or even discover unknown sequence variations without designing additional specific probe assays,” explains Justin Barker, product manager for qPCR Systems at Bio-Rad Laboratories. Melt analysis also has the ability to have more multiplex density for variants in the same region than can be addressed with probes due to less optimization and spectral constraints.

In addition to characterizing nucleic acid samples based on their dissociation behavior and detecting small differences in PCR amplified sequences, samples can be discriminated according to their sequence length, GC content, and strand complementarity. Shonali Paul, COO at PREMIER Biosoft, adds that HRMA is a quick and cost-effective approach that reduces the need to design and purchase multiple pairs of primers and probes, detecting a single base substitution with only a very subtle influence on the melting temperature.

The melting temperature at which two DNA strands of an amplicon come apart depends on the DNA sequence. While samples from two different people should give the same shaped melt curve, any mutation in the amplified DNA of one person will alter the melting temperature of those DNA strands and so will result in a different melting curve. In cases of allelic variants for example, each variant generates a slightly different melting curve. “Sequence identity means melting curve identity,” explains Wittwer. “If a melting curve changes, it directly correlates with a change in the sample sequence.”

What HRMA can do

As a universal technology applicable in a multitude of research domains including biomarker discovery and methylation studies, HRM applications cover single nucleotide variation (SNV) analysis, mutation detection (including point mutations and indels), determination of methylation status at a genetic locus, short tandem repeat (STR) analysis, and characterization of similar but not identical pathogens. HRMA can also be used to determine the homo and heterozygosity of a gene at a particular locus. Changes in enzyme restriction or electrophoretic and chromatographic profiles can even be measured, reducing analysis time and risk of contamination.

One of the most frequent applications is SNP genotyping, notes Wittwer. Barker sees many customers performing SNP genotyping applications using Precision Melt software due to its easy clustering and flexibility in scale. The software enables effortless interrogation of multiple experiments per run using well group designation and the ability to analyze multiple genotyping plates together, applying automated cluster plotting in either case.

“Precision Melt's greatest value is allowing for applications where sequences do not have to be known and probes designed. In this way, gene scanning for sequence variants can still be performed for identifying genomic variants in specific regions for labs where NGS has not been utilized. Once validated, consistent detection assays can be designed for those small variants with high-resolution melt,” says Barker. He comments that HRMA is now being used in CRISPR screening, being well-adapted to identify edited clones for confirmation sequencing. This can be performed cheaper and easier than sequencing technologies alone and more effectively then cumbersome enzymatic surveyor assays.

Advantages of HRMA

  • HRMA provides significantly more information, down to single nucleotide differences, in less than an hour and at much lower costs, than alternative methods for the discovery, detection, and/or comparison of small or unknown sequence variations.
  • It is easy to use and flexible enough to be used in various applications.
  • HRMA characterizes nucleic acid samples based on their dissociation behavior and detects small differences in PCR amplified sequences, as well as discriminates samples according to their sequence length, GC content, and strand complementarity.
  • HRMA is a quick and cost-effective approach that reduces the need to design and purchase multiple pairs of primers and probes, providing a simple workflow, fast and accurate processing, and low reagent consumption.
  • Advances in fluorescent dyes, instruments, and software have enabled a versatility in newly developed HRM methods for variant scanning and genotyping that has set it apart from alternative approaches.

HRMA is also common in epigenetic investigations where modifications effect melting affinity, such as methylation. Methylated DNA can be treated by bi-sulphite modification that converts non-methylated cytosines to uracil. The amplicons resulting from the unmethylated DNA will have a lower melting point than the amplicons derived from methylated DNA. “Using HRMA, the proportion of methylation in a given sample can be determined by comparing it to a standard curve generated by mixing different ratios of methylated and non-methylated DNA together. This further helps to unveil the information of the character of a tumor, for example, and how far it deviates from the normal state,” adds Paul.

Supporting HRM with new targeted technologies

The tools used to perform HRMA can influence quality of results. “HRM analysis can be optimized from standard melt curve analysis by using brighter dyes at higher concentrations, instruments with precise temperature control to collect fluorescence data, and more sophisticated software to plot high-density HRM data,” says Paul.

Certain dyes are more useful than others, Wittwer suggests, such as LCGreen (BioFire) and SYTO9 (Thermo Fisher) that better detect heteroduplexes for enhanced sensitivity. HRM performance can be advanced further with reliable template purity, high specificity HRM PCR kits, an HRM instrument with superior temperature uniformity and powerful software packages for any kind of HRM data analysis, notes Missel. QIAGEN has been developing HRM-focused tools since its discovery, enabling users to streamline the entire HRM process by offering dedicated solutions for each of these working steps.

QIAGEN kits for the preparation of a gDNA template from any sample type allow the manual or automated isolation of pure starting material. Dedicated HRM kits provide highly specific PCR amplicons, which are important for unambiguous HRM results. Due to its unique design, the Rotor-Gene Q thermocycler guarantees minimal thermal variability from sample-to-sample with a temperature uniformity of +/- 0.01 °C. And finally, QIAGEN’s HRM analysis software ScreenClust represents an advanced tool for the statistical analysis of HRM results.

For SNP genotyping, PREMIER Biosoft’s Beacon Designer is a commercially available software that facilitates the design of highly specific and efficient HRM primers. It designs optimal SNP flanking primers to generate the shortest possible amplicon. As soon as the HRMA primer search is complete, Beacon Designer displays the search quality status and reports detailed results, including both the sense and antisense primer rating, primer sequence, position, length, melting temperature, percent GC, maximum hairpin dG, maximum self-dimer dG, repeat and run length, optimum annealing temperature, maximum cross dimer dG, product length, and product melting temperature (both wild and mutant).

Paul adds that Beacon Designer is also equipped with BLAST search-like features that allow the user to perform BLAST searches against NCBI for every target sequence and primer pair. Following the BLAST search, a template secondary structure search can be performed to avoid regions of secondary structure while designing primers.

Advances in fluorescent dyes, instruments, and software have enabled a versatility in newly developed HRM methods for variant scanning and genotyping that has set it apart from alternative approaches, such as sequencing and Taqman that require more time and materials with additional separations or labeled probes. With a simple workflow, fast and accurate processing, and low reagent consumption, HRMA has proven to be a method of choice across diverse applications in labs studying nucleic acids.