Featured Article
Wednesday June 23, 2010
by Caitlin Smith
High resolution melt (HRM) analysis is one of those “why didn’t I think of that?” special twists on basic nucleotide behavior during
PCR, in which melting curves are compared to detect sequence differences. HRM analysis is seeing a boom of interest recently. “HRM is emerging so rapidly that it is difficult to rank the most exciting developments,” says Ulla Deutsch, senior global product manager for amplification at
Qiagen, who notes that mutation scanning and SNP genotyping are among the main applications of HRM, with newer applications including pathogen typing. Arun Apte, media contact at
Premier Biosoft, says that “with rapid improvements in resolution capabilities of the melting instruments, HRM analysis can further be applied for multiplex SNP genotyping, gene scanning for mutation identification, methylation studies, quantification of gene dosage, establishing phylogeny and many more sequence based studies.” Here is a look at some new uses and methods for HRM.
Increasing use and increasing innovation
One challenge to advancing HRM analysis today is “the gap between customer expectations of performance and the actual error rate observed in HRM experiments,” says Gordon Janaway, product manager for SYBR Green and HRM qPCR reagents at
Life Technologies. “Some of this error is the result of poorly designed HRM systems, but in other instances (for example, A/T SNP mutations) the melt profiles of amplicons can be almost identical, and samples cannot be distinguished by melt curve alone. For example, HRM is an effective solution for scanning amplicons with unknown mutations, where discrimination between homozygous wild-type and heterozygous samples is required. However, HRM can be less reliable for routine genotyping of known SNP mutations due to very small differences in Tm between homozygous samples.”
Janaway notes that an especially interesting new use of HRM is the analysis of “bisulfite-converted DNA to determine the methylation status of genomic DNA samples. Depending on the experimental design, HRM can be used to either interrogate specific CpG sites for methylation, or estimate the percent methylation of CpG sites across a target genomic region.” Life Technologies’ complement of HRM tools includes instrumentation, reagents, and software for Applied Biosystems’ 7500-Fast and
7900 Real-Time PCR Systems. Recently Applied Biosystems introduced its MeltDoctor™ HRM Reagents and the ViiA™ 7 Real-Time PCR system, also HRM-compatible.
Qiagen also offers two new HRM tools—the Type-it® HRM PCR Kit and Rotor-Gene® ScreenClust HRM Software. The former detects gene mutations and SNPs without requiring optimization of new HRM assays. The latter “overcomes the constraints of current HRM software solutions such as manual data interpretation by offering a stringent statistical approach to extract, process and group sample characteristics in HRM data sets automatically,” says Deutsch. “This approach even allows genotyping without control samples. In combination with the Rotor-Gene Q or Rotor-Gene 6000 instruments, researchers are able to differentiate between alleles in less time with higher significance.”
Oligo design for HRM
Beacon Designer is currently the only tool to design oligos specifically for HRM analysis, according to Apte. “The program employs a proprietary algorithm that enables designing the best HRM analysis primers for detecting SNP classes I and II for heterozygotes and homozygotes,” he says. “The primers are designed flanking a SNP or mutation of interest to generate the shortest possible amplicons with detectable melting temperature variation, taking into consideration all the parameters essential for HRM analysis. Beacon Designer enables the use of pre-designed or published forward or reverse primers by analyzing the oligo properties for a given assay and then designing a complimentary oligo.”
HRM analysis is increasingly being used for pathogen identification. “This requires the design of HRM probes that can uniquely identify any member of a closely related set of sequences,” says Apte. “We have added HRM support to our pathogen identification tool, AlleleID. This enables a researcher to take a set of closely related bacterial or viral sequences, perform an alignment, and AlleleID will design probes that will uniquely identify any member of the set.”
Look, no labels
Genotyping using unlabeled probes is another promising development in the evolution of HRM techniques, according to Carl Fisher, product manager for PCR at
Bio-Rad. “While it is straightforward to distinguish between homoduplexes and heteroduplexes, determining precisely which homozygote is present can be quite challenging using HRM if there is little difference in the Tm,” says Fisher. “Adding an unlabeled probe (blocked at the 3’ end to prevent extension), that is specific for the SNP of interest, will result in a corresponding melt peak appearing earlier than that of the full length amplicon if the targeted SNP is present in that sample. Additionally, any mismatches are more easily detected due to the much smaller size of the probe sequence relative to that of the amplicon.”
Bio-Rad’s newest HRM offerings include their Precision Melt Analysis software for use with their
CFX96 and
CFX384 real-time PCR detection systems. “This software has a user-friendly interface with a workflow that simplifies data analysis,” says Fisher. “Some of the new features include easily customizable analysis settings, a multiple data view that allows users to manually assign sample genotypes, analysis of multiple experiments from a single plate, and the ability to analyze multiple data files (for melt studies).”
Fisher believes that one of the biggest challenges to advancing HRM analysis “is people’s perception that HRM is difficult to perform and that it requires a lot of extra time and effort,” he says. “In actuality, HRM is an extension of the regular melt curves that users have been performing for many years now to analyze their
qPCR amplification products, but with smaller temperature increments and using saturation dyes. Assay optimization is an area that can be neglected, but it is particularly important for HRM. A little extra effort spent on optimization can greatly reduce frustration at later stages of analysis.”
At
Roche Diagnostics, HRM is handily accomplished with their LightCycler® 480 Real-Time PCR instrument. “[One] of the advantages it offers is a therma-base between the Peltier and the block, allowing very even temperature distribution across the block, giving you very even melting,” explains Larson Manifold, marketing manager in applied science at Roche Diagnostics. “Of course, that’s very important for this application. It also does a continuous melting, assessing the fluorescence throughout this continuous melt, unlike some instruments which step up as they melt, stepping up maybe ½ to ¼ of a degree, to hold that temperature and then acquire.” Roche also has a new
master mix using a new fluorescent dye for detecting double-stranded DNA, called
LightCycler® 480 ResoLight Dye. “This is a saturating, intercalating dye [that’s] less inhibitory to PCR [and more temperature stable],” says Manifold.
Manifold describes one of the conundrums of HRM analysis: “You want to do large PCR to get the most out of a single run, but the larger your amplicon gets, the less impact on the melting curve these small changes make,” he says. “So you have a point of diminishing returns. Ideally, we want to see these amplicons be around 300 bp or less, but 500 bp is really what we call the realistic limit to give us good confidence in anything. We’ve seen people go up to a thousand and still get decent results, but there is a possibility of missing something.” But scientists are developing strategies to meet this challenge. “Unlabeled probes are a nice one because that’ll allow us to get absolutely reliable information on an area that we want to focus on, while getting general information about the rest of a large amplicon,” says Manifold. “Even though we might miss something, we’ve got our specific information that we wanted from the unlabeled probes. So there are strategies, but there are still limitations.”