HRM - High Resoltution Melt (1)
HRM page (2)
HRM page (3)
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.
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.
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
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.
HRM data normalizationshape & shift
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).
HRM software application
High Resolution Melting (HRM) Software v 2.0 by Applied Biosystems
The Applied Biosystems HRM Software provides an easy and intuitive workflow that:
HRM Workflow in the LC 480
Gene Scanning by High Resolution Melting Curve Analysis generally requires the use of
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:
High-resolution melting curve analysis on the LightCycler 480 PCR system (presented by Roche Aplied Science)
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.
The LightCycler® 480 real-time PCR system: a versatile platform for genetic variation research
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.
Transfering PCRs to HRM-assays on the LightCycler 480 System- Examples for BRCA1
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.
Rapid high-throughput Methylation analysis using the LightCycler 480 system (presented by Roche Aplied Science)
Microsatellite Analysis of Grapevine Varieties by HRM Analysis (by John Mackay)
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.
High Resolution Melt - TALKs: