HRM Introduction

 
The most important High Resolution Melting application is gene scanning - the search for the presence of unknown variations in PCR amplicons prior to or as an alternative to sequencing. Mutations in PCR products are detectable by High Resolution Melting because they change the shape of DNA melting curves. A combination of new-generation DNA dyes, high-end instrumentation and sophisticated analysis software allows to detect these changes and to derive information about the underlying sequence constellation.

With HRM, these and other applications are done using low-cost generic dyes where previously custom labeled probes such as TaqMan® or fluorescence resonance energy transfer (FRET) probes were required. HRM is thus a simpler and much more cost-effective way to characterize samples.

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In molecular biology High Resolution Melt or HRM analysis as it will be referred to herein is a hugely powerful technique for the detection of mutations, polymorphisms and epigenetic differences in double stranded DNA samples. It has advantages over other genotyping technologies. Namely:

• It is massively cost effective vs. other genotyping technologies such as sequencing and Taqman SNP typing. This makes it ideal for large scale genotyping projects.
• It is fast and powerful thus able to accurately genotype huge numbers of samples in rapid time.
• It is simple. With a good quality HRM assay powerful genotyping can be performed by non-geneticists in any laboratory with access to an HRM capable real-time PCR machine.
• http://en.wikipedia.org/wiki/High_Resolution_Melt

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For several years, various researchers and instrument makers have independently investigated the utility of high-resolution DNA dissociation analysis. For example, the team at Idaho Technology has done an admirable job of vigorously promoting their research through traditional journal publications. Conversely, Corbett Life Science does not pursue publication, but instead relies on the publications of customers to promote the technology. Regardless, both companies have independently advanced the field of high resolution dissociation analysis and successfully introduced what has now become known as high resolution melt (HRM) analysis.
 
Idaho Technology was first to market with an instrument made specifically to do dissociation analysis; the HR-1. The HR-1 was a showpiece for the technology with the singular aim of producing the most detailed melt curve possible. As such, it opened the eyes of many to the potential of HRM and remains the performance benchmark for the acquisition of an individual melt curve. However the HR-1 is not capable of thermal cycling and can only analyze a single sample from within a glass capillary per run making data analysis time consuming. http://www.idahotech.com/HR-1/index.html
                                  
Multi-well instruments with greater practical utility were introduced to the market very soon after the HR-1. The first multi-well HRM instruments were the Rotor-Gene 6000 (Corbett Life Science) and the LightScanner (Idaho Technology) (PDF). These two instruments were introduced at about the same time but employed fundamentally different technical innovations to achieve HRM. The LightScanner uses a modified block-based design available in 96-well or 384-well versions. Despite advanced engineering, it still suffers from measurable sample-to-sample thermal and optical variation and is unable to match the performance benchmark set by the original HR-1 instrument. Like the HR-1, the LightScanner is not capable of thermal cycling.
 
The Rotor-Gene 6000 was the first of the multi-well instruments capable of both thermal cycling and HRM. This dual capability enables samples to be fully processed in the one instrument (i.e. pre-amplification and HRM done consecutively in the one run). A major advantage of this is that amplification plots can be used to help interpret HRM results since aberrant amplification plots (i.e. those that amplified differently to what was expected) also produce aberrant HRM data. In this way compromised samples can be easily identified and removed from downstream HRM analysis. The main advantage of the Rotor-Gene for HRM stems from its rotary design, in which samples spin under centrifugal force past a common optical detector. This is seemingly ideal for HRM as thermal or optical variation between samples is insignificant. The result is that the Rotor-Gene HRM performance closely matches the HR-1 benchmark with the compromise that samples are not arranged in a conventional array format (as they are in block-based instruments) but are instead arranged around the perimeter of a spinning rotor.
                                                                                                                                                                                      
The more recently introduced LightCycler 480 (Roche Molecular Systems) is capable of HRM and thermal cycling. The LightCycler 480 is a block-based instrument design and it has better thermal uniformity than other block-based instruments, it nevertheless does exhibit measurable thermal and optical non-uniformity.
                                                                                                                                                                                          
Other instrument providers are now rushing to introduce HRM capability and some are planning to release software upgrades to support HRM analysis. The danger here is that instruments not specifically engineered for HRM will deviate so much from the HR-1 performance benchmark that careful investigation will need be done before accepting those instruments as HRM capable.

	Example HRM data for each of the multi-well HRM systems discussed here is shown in the figures (A-E) below. For comparison purposes, similar data for two standard real-time PCR instruments (i.e. not engineered for HRM) is also shown. All data has been enlarged without modification directly from (Herrmann et al 2007) Normalized melting curves of a 110 bp beta-globin amplicon (triplicate HRM data) containing single and double SNPs are shown.
	A: Rotor-Gene: all four genotypes are clearly distinguished (click figure to enlarge)
	B:  LightScanner: only heterozygotes can be distinguished (PDF) (click figure to enlarge)
	C: LightCycler 480: double heterozygote can be clearly distinguished (click figure to enlarge)
	D: AB 7300: double heterozygotes can be distinguished (click figure to enlarge)
	E: MasterCycler: none of the genotypes can be distinguished (click figure to enlarge)

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Before HRM curves are plotted, the raw data is first normalized. Melt curves are normally plotted with fluorescence on the Y axis and temperature on the X axis. This is similar to real-time PCR amplification plots but with the substitution of temperature for cycle number. As with real-time PCR plots, the fluorescence axis of HRM plots is normalized onto a 0 to 100% scale.
 
An emerging trend is to also apply normalization to the temperature (X) axis. This has the desired effect of compensating for well-to-well temperature measurement variations between samples. Known as “temperature shifting”, it was introduced by Idaho Technology and is now also supported by the Roche LightCycler 480. Unfortunately, temperature shifting normalization removes any potential discriminatory power provided by the temperature data.
 
For some applications, temperature shifting normalization may be a useful solution but for many routine applications it is actually detrimental. A good example of this is the discrimination of homozygous SNPs. On the one hand, heterozygous samples are often more easily discriminated after temperature shifting normalization (because their curves have a complex shape), but the discrimination of homozygous samples is usually made more difficult because they often have a simple and identical curve shape (Figure 1). While homozygous SNP samples have an identical curve shape, they can usually be discriminated by HRM analysis by observing a change in their respective Tm’s. This characteristic means the melt plots of different homozygotes will be offset one from another thereby allowing them to be readily discriminated (so long as temperature shifting normalization is not applied and the HRM temperature data is precise enough). Currently, the only instrument system that does not use temperature shifting normalization and can reliably discriminate homozygous SNPs is the Rotor-Gene (Corbett Life Science). The Rotor-Gene can discriminate homozygotes because well-to-well thermal variation is so low on that instrument that the collected temperature data is sufficiently precise (Figure 2).

	Figure 1: Thermal shifting normalization on the LightCycler 480 (Roche Applied Science) Triplicate HRM data was captured on a Roche LightCycler 480 for SNP genotyping (Herrmann et al 2007)  Normalized melting curves are of a 110 bp beta-globin amplicon. Genotypes are discriminated by color as follows; green = homozygous wild type, red = homozygous mutant (20A>T), black = single heterozygous mutant (20A>T), blue = double heterozygous mutant [9C>T; 20A>T]. Plots are shown before (A) and after (B) temperature shifting normalization. Double normalized melt curves of homozygous genotypes overlay and cannot be discriminated; however, discrimination of heterozygous genotypes is improved.
	Figure 2: Thermal sifting normalization on the Rotor-Gene (Corbett Life Science) Triplicate HRM data was captured on a Rotor-Gene for SNP genotyping (Herrmann et al 2007) Normalized melting curves are of a 110 bp beta-globin amplicon. Each category of SNP genotype can be readily discriminated prior to thermal shifting normalization. However, when curves are thermal shifted the homozygous genotypes overlay precisely and can no longer be discriminated.

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High-resolution DNA Melting Analysis When it comes to genotyping and mutation scanning, high-resolution DNA melting is emerging as the technique of choice because it is inexpensive simple, accurate and rapid.  Development of this method of DNA analysis has been underway since its introduction in 2002 by a team of researchers from our Pathology Department led by Dr. Carl Wittwer and Dr. Karl Voelkerding at the University of Utah coupled with collaborative efforts from Idaho Technology. High-resolution melting required new instrumentation.  The first high-resolution instrument developed, named the HR-1, remains the most accurate with the fastest analysis speed, while the LightScanner has the highest throughput. In addition to the special instrumentation, high-resolution melting uses special saturation dyes that fluoresce only in the presence of double stranded DNA.  These dyes are included in the PCR amplification process.  When the sample is heated to high temperatures, the DNA denatures and the fluorescent color fades away as the double stranded DNA separates, generating a melting curve. Because different genetic sequences melt at slightly different rates, they can be viewed, compared, and detected using these curves.  Even a single base change will cause differences in the melting curve.  The process can be used for specific genotyping, comparing sequence identity between two DNA samples, and scanning for any sequence variant between two primers. High-resolution DNA melting is becoming more popular as its accuracy and simplicity is recognized.  High-res DNA melting makes it possible to quickly and accurately determine whether DNA sequences match, providing an interesting option for transplantation matching and forensics. Genotyping via high-resolution melting is more streamlined and less expensive than methods that use complex probes.		No processing is required, and when combined with the speed of rapid-cylce PCR, has interesting potential for personal DNA diagnostics. For example, the amount of medication a person needs is often dependent on sequence variants in genes that can be determined through high-resolution DNA melting. Hi-res melting can also be used to scan large genes for variation, in many cases greatly reducing or eliminating the need for sequencing. Although high-resolution DNA melting is relatively new, it is expanding and being improved upon by our talented team of scientists in Pathology and we are excited to be at the forefront of such innovative and important technology.
More information at  http://www.path.utah.edu/news/hi-res-dna-melting-analysis

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HRM software application
High Resolution Melting (HRM) Software v 2.0  by Applied Biosystems

• No Temperature Shift Required.
• Fast. Accurate.
• Identify more new variants, quickly and accurately.
• High Resolution Melting (HRM) analysis is an alternative to dHPLC sequencing screening of new gene variants. The HRM Software is now available on the Applied Biosystems 7500 Fast System and on the 7900HT Fast Real-Time PCR System. The 7500 Fast Real-Time PCR System delivers precise results with fast thermal cycling in a standard 96-well format. Achieve high-throughput HRM analysis with the 384-well 7900HT, the gold standard high throughput system. The AB HRM application does not require temperature shifting, which results in a greater likelihood of identifying new homozygous mutations than methods that require temperature shifting.

The Applied Biosystems HRM Software provides an easy and intuitive workflow that:

• Shortens analysis time by auto-calling genotypes and automatically omitting the no template controls
• Minimizes subjective analysis by automatically grouping unknown variant clusters
• Allows easy data review with customizable plot views, expandable windows, and one-click color assignment to highlight curves of interest
• Ability to analyze multiple targets (assays) on one plate

    => download the HRM guide

A.   7500 Fast Real-Time PCR System	B.   7900HT Fast Real-Time PCR System
Heterozygote    Homozygous - Wildtype    Homozygous - Variant Figure 1 Difference plot generated with the Applied Biosystems HRM application on the 7500 Fast & 7900HT Fast Real-Time PCR Systems.
Figure 2  Difference plot generated on another plate-based real-time HRM system.	The ability to easily identify new variants is key for successful HRM applications. By eliminating the temperature shift step, the Applied Biosystems HRM solution (Fig 1) was able to clearly distinguish homozygous variant samples from homozygous wildtype samples in 97.5% of the population, whereas the other HRM system from Competitor R (Fig 2) was only able to distinguish them in 10% of the population. All genotypes were auto-called by the respective software packages and were not altered by the operator. Class 1 SNP (A/G), multiple technical replicates of nine DNA samples representing three genotypes: homozygous wildtype (G/G), homozygous mutant (A/A) and heterozygous (A/G).

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Gene Scanning by High Resolution Melting Curve Analysis generally requires the use of

• a special generic DNA dye that works at high, saturating concentrations without inhibiting PCR and therefore leads to homogeneous staining of homo-or heteroduplex DNA
  
• an instrument with suitable excitation/emmission wavelengths, high data acquisition rates, and outstanding temperature homogeneity
  
• a software algorithm that analyzes the shape of the melting curves and groups those that are similar.

In a Gene Scanning experiment, sample DNA is first amplified via real-time PCR in the presence of a proprietary saturating DNA dye. A melting curve is then performed using high data acquisition rates, and data are finally analyzed using a Gene Scanning Software, by three basic steps:

1. Normalization: the pre-melt (initial fluorescence) and post-melt (final fluorescence) signals of all samples are set to uniform, relative values from 100% to 0%
2. Temperature shifting: the temperature axis of the normalized melting curves is shifted to the point where the entire double-stranded DNA is completely denatured. Samples with heterozygous SNPs can then be easily be distinguished from the wild type by the different shapes of their melting curves. 
3. Difference Plot: the differences in melting curve shape are further analyzed by subtracting the curves from a reference curve. This helps cluster samples automatically into groups that have similar melting curves (e.g., those who are heterozygote as opposed to homozygotes).
4. Application Manuals and Technical Guidelines
   LightCycler® 480 Technical Note No. 1: "High Resolution Melting: Optimization Strategies" (12 pages)
   Enables HRM users to successfully set up and carry out mutation scanning experiments
   Copy available for download => LC480-Technical-Note-01-HRM.pdf
   

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