HRM Introduction High Resolution Melting ( HRM) is a novel, homogeneous, close-tube, post-PCR method, enabling genomic researchers to analyze genetic variations (SNPs, mutations, methylations) in PCR amplicons. It goes beyond the power of classical melting curve analysis by allowing to study the thermal denaturation of a double-stranded DNA in much more detail and with much higher information yield than ever before. HRM characterizes nucleic acid samples based on their disassociation (melting) behavior.
Samples can be discriminated according to their sequence, length, GC content or strand complementarity. Even single base changes such as SNPs (single nucleotide polymorphisms) can be readily identified. HRM Applications The introduction of HRM has renewed interest in the utility of DNA melting for a wide range of uses, including:. Mutation discovery (gene scanning).
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Screening for loss of heterozygosity. DNA fingerprinting. SNP genotyping.
Characterization of haplotype blocks. DNA methylation analysis. DNA mapping. Species identification. Somatic acquired mutation ratios. HLA compatibility typing. Association (case/control) studies.
Allelic prevalence in a population. Identification of candidate predisposition genes. 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.
HRM Instrumentation 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. 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 and the 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 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 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. 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 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. There are two ways HRM curve plots can discriminate between samples; by “ Shape”, i.e.
Using detail in the shape of the melt curve itself and by “Shift”; i.e. The thermal offset of a curve from other curves. 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 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 (20AT), black = single heterozygous mutant (20AT), blue = double heterozygous mutant 9CT; 20AT. 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).
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 HRM software application High Resolution Melting (HRM) Software v 2.0 by Applied Biosystems. No Temperature Shift Required. 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 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).
HRM Workflow in the LC 480 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:. 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%. 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. 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). 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. Roche Applied Science´s LightCycler® family of real-time PCR systems offer fast, accurate and versatile platforms for genetic variation research. The new plate-based LightCycler® 480 System provides the temperature homogeneity and optical characteristics required for high-performance melting-curve analysis (MCA). On the level of data acquisition and available detection channels, this new instrument opens the way to more advanced applications in the emerging field of gene scanning where amplicons can be screened for unknown sequence variations with low efforts in time and cost.
Real-time PCR is a well established technique for studying genetic variation using various probe-based methods for genotyping as well as high-resolution analysis of whole amplicons melted in the presence of saturating DNA dyes. The latter, relatively new, method allows screening for unknown mutations or DNA modifications. The LightCycler® 480 real-time PCR system is a multiwell plate–based instrument that provides integrated applications for detecting and characterizing genetic variation using all these methodological approaches. High-resolution melting curve analysis (hrMCA) is an attractive technique to scan for unknown mutations in genes. To evaluate how easy or difficult it is to design hrMCA assays using the LightCycler® 480 Instrument, we selected 3 different fragments in exon 11 of the BRCA1 gene, designed an MCA assay, and tested its sensitivity to detect known variants. (by Corbett Life Science) A very good explanation of the HRM method! (by Corbett Life Science) (by Qiagen) Recently, HRM was the subject of a detailed and independent Technology Assessment report from the National Genetics Reference Laboratory (Wessex, UK).
A wide range of sample types were tested, including examples of challenging G to C and A to T single base substitutions. The full report is now available for download = Mutation Scanning by High Resolution Melt Analysis.
Evaluation of Rotor-Gene 6000 (Corbett Life Science), HR-1 and 384-well LightScanner (Idaho Technology). Guidelines for Developing Robust and Reproducible High-Resolution Melt Analysis Assays by Sean Taylor, Rachel Scott, Richard Kurtz, Viresh Patel, and Frank Bizouarn - Bio-Rad Laboratories Classifying and understanding genetic variation between populations and individuals is an important aim in the field of genomics. Many common diseases (diabetes, cancer, osteoporosis, etc.) and clinically relevant phenotypic traits are elicited from the complex interaction between a subset of multiple gene products and environmental factors. High resolution melt (HRM) analysis is the quantitative analysis of the melt curve of a DNA fragment following amplification by PCR and can be considered the next-generation application of amplicon melting analysis. It is a low-cost, readily accessible technique that merely requires a real-time PCR detection system with excellent thermal stability and sensitivity and HRM-dedicated software. However, careful sample preparation and planning of experimental and assay design are crucial for robust and reproducible results. The following guidelines assist in the development of such assays.
Unique rotary design for outstanding performance The unique centrifugal rotary design of the Rotor-Gene Q makes it the most precise and versatile real-time PCR cycler currently available (see figure 'Cross-section of the Rotor-Gene Q'). Each tube spins in a chamber of moving air, keeping all samples at precisely the same temperature during rapid thermal cycling. Detection is similarly uniform. When each tube aligns with the detection optics, the sample is illuminated and the fluorescent signal is rapidly collected from a single, short optical pathway. This thermal and optical uniformity results in sensitive, precise, and fast real-time PCR analysis (see figure 'Precise real-time PCR analysis').
It also eliminates sample-to-sample variations and edge effects. These are unavoidable in traditional block-based instruments due to temperature gradients across the block and multiple, complex optical pathways. The rotary design delivers:. Well-to-well variation ±0.02°C. Uniform detection eliminating the need for ROX reference dye. Fast ramping and negligible equilibration times for short run times.
Complete confidence in your results! Unrivaled optical range enables multiple applications Whether your assay is based on intercalating dyes such as SYBR Green, probes such as hydrolysis (TaqMan), hybridization (FRET), Scorpion probes, or other multiplex chemistries, the Rotor-Gene Q meets your requirements.
With up to 6 channels spanning UV to infrared wavelengths, the cycler delivers the widest optical range currently available (see Table 'Channels for optical detection'). In addition, the software allows you to create new excitation/detection wavelength combinations, which means that the Rotor-Gene Q is compatible with dyes you may use in the future. Expand your research with HRM High-resolution melting analysis (HRM) is a closed-tube, post-PCR analysis that has raised enormous scientific interest. HRM characterizes double-stranded PCR products based on their dissociation (melting) behavior. It is similar to classical melting curve analysis, but provides far more information for a wider range of applications. PCR products can be discriminated according to sequence, length, GC content, or strand complementarity, down to single base-pair changes. Previously unknown and even complex sequence variations can be readily detected and characterized in a robust and straightforward way.
The rotary design of the Rotor-Gene Q and its outstanding thermal and optical performance are highly suited to HRM. The HRM option for the Rotor-Gene Q includes:.
A specially tuned high-intensity optical HRM channel. Thermal resolution down to 0.02°C. High data acquisition rates. Comprehensive HRM software The Rotor-Gene Q is the only real-time cycler currently capable of deciphering the most difficult class IV SNPs by HRM. Harness the power of HRM using dedicated QIAGEN HRM Kits for applications such as genotyping (see figure 'HRM analysis of a class IV SNP with less than 0.1˚C difference between homozygote alleles' for data from the Type-it HRM PCR Kit), quantitative methylation analysis (see figure 'Highly sensitive results with detection of even low percentages of methylated DNA' for data from the EpiTect HRM PCR Kit), gene scanning, and sequence matching.
The Type-it HRM PCR Kit reliably and accurately detects gene mutations and SNPs. The EpiTect HRM PCR Kit enables fast screening and accurate detection of changes in CpG methylation status of bisulfite converted DNA. Superior new software available for genotyping and mutation detection using HRM analysis Rotor-Gene ScreenClust HRM Software is the most powerful tool currently available for analysis of HRM data from the Rotor-Gene Q or Rotor-Gene 6000 cycler.
Rotor-Gene ScreenClust HRM Software is an extension to the Rotor-Gene operating software. By grouping samples into clusters, Rotor-Gene ScreenClust HRM Software opens a new dimension in HRM analysis for applications such as genotyping and mutation screening.
Flexible formats match your workflows The Rotor-Gene Q supports multiple PCR tube formats to suit a range of needs. Changing the format, by simply switching the snap-fit metal rotor that holds the tubes, takes just seconds. As well as tubes, Rotor-Discs are available, which offer accelerated setup and higher throughput. Rotor-Discs are circular plates of vertically oriented reaction wells. The Rotor-Disc 100 is the equivalent of a 96-well plate with an additional 4 reference wells. These extra wells can be conveniently used for more reactions or additional controls.
Alternatively, the Rotor-Disc 72 has 72 wells. Rotor-Discs can be quickly and easily sealed with plastic film using a Rotor-Disc Heat Sealer. For all you need to run reactions using Rotor-Discs, choose the Rotor-Disc 100 Starter Kit or the Rotor-Disc 72 Starter Kit. You can perform manual reaction setup, or take advantage of QIAGEN's automated solutions for reaction setup. The QIAgility is cost-effective and delivers rapid, high-precision PCR setup, while the QIAsymphony AS is ideal for laboratories performing routine PCR tests on a day-to-day basis. Both instruments perform automated reaction setup in Rotor-Gene formats, allow direct transfer of sample lists, and are supplied with verified protocols for real-time PCR master mixes. Software enables quantification and enhances data security The comprehensive Rotor-Gene Q software package supports all current state-of-the art real-time analysis procedures from basic to advanced algorithms.
This provides complete freedom to analyze your valuable experimental data and increases the reliability of your results. Data security is assured and all process steps are trackable from starting the run to exporting the results.
See figure 'Analysis procedures supported by Rotor-Gene Q software'. (For highly sophisticated analysis of HRM data, an extension to the Rotor-Gene operating software is available: Rotor-Gene ScreenClust HRM Software).
Minimum maintenance, maximum convenience The Rotor-Gene Q is engineered to reduce the need for maintenance and to maximize ease of use. This saves time and costs and allows you to focus on your research, not on keeping the cycler up and running. Convenient features of the Rotor-Gene Q include:. Lifetime guarantee on highly stable LEDs, no expensive lamps or lasers to change, no gradual performance loss of light source.
No optical calibration needed at installation or when the instrument is moved. No sample block to clean. No condensation or bubbles in reactions due to rotation. Small, light, and robust, simply place the instrument wherever you like!
Easy routine verification Laboratories may often want to verify thermal accuracy. For most cyclers, this requires interaction with a service engineer. With the Rotor-Gene Q, this is not necessary. Instead, the easy-to-use, cost-effective automates accuracy testing. The full procedure takes only a couple of minutes.
Reliable support for your peace of mind In the unlikely event of any service issues with your Rotor-Gene Q, QIAGEN Instrument Service provides comprehensive support services to ensure the continued success of your PCR applications. Features: Dynamic range: 12 orders of magnitude. Kits designed for this instrument: artus QS-RGQ Kits (not available in all countries), RG SYBR Green PCR Kits; RG SYBR Green RT-PCR Kit; RG Probe PCR Kits; RG Probe RT-PCR Kit; RG Multiplex PCR Kit. Optical System: Up to 6 channels spanning UV to infra-red wavelengths; Excitation sources: High energy light-emitting diodes; Detector: Photomultiplier; Acquisition time: 4 s.