What is HRM? HRM greatly extends the usefulness of real-time methodology from simple PCR-based sequence detection and quantification to now encompass detailed post-PCR sequence analysis. HRM enables a wide range of new applications not possible using conventional melting or probe-based detection methods. For example, it allows previously unknown and even complex sequence variations to be readily detected and characterized in a simple and straightforward way. Although new, HRM is fast becoming a mainstream tool for molecular analysis having already found diverse application (see below).
HRM is a closed-tube, post-amplification method done that uses simple and inexpensive generic dye chemistry. While essentially similar to conventional DNA melt analysis (i.e. the monitoring of strand dissociation behavior), it has greatly extended utility because it can capture so much more detail. Key to HRM is a new generation of purpose-built instrumentation that provides acute thermal and optical precision, high speed data acquisition, and new software tools for data analysis. HRM characterizes nucleic acid samples according to their sequence, length, GC content, or strand complementarity. Even single base changes such as SNPs (including challenging Class IV SNPs) can be readily identified, as shown opposite.
Why use HRM? Allele-specific primers or probes are not needed for HRM as the same dye can be used for all targets. This means less sequence information is needed to design new assays. It also saves the time and effort required to design, order, aliquot, and store expensive custom fluorogenic probe(s). Besides its simplicity and convenience, HRM offers greater economy and higher throughput when screening for novel mutations or complex sequence variations compared to traditional methods such as dHPLC and sequencing.
HRM exploits a robust biophysical measurement to produce information-rich data. In addition to the melt data for a particular sample, its corresponding real-time amplification data can also be used to provide valuable insight into sample quality and reaction efficacy. In this way abnormal amplification plots are used to identify and remove failed reactions from the analysis so they don't obscure results. HRM thus has advantages over methods that acquire information from just the amplification process.
Chemistry HRM is normally done immediately after amplification, so reaction setup is the same as for a regular real-time reaction, the only change being the addition of a DNA intercalation dye. The dyes SYBR® Green 1, SYTO®9, EvaGreen™, LC Green®, and LC Green® Plus have all been used successfully. The same dye monitors both the upfront real-time amplification reaction as well as the subsequent HRM.
Instrumentation Recent advances in instrumentation has made HRM possible. HRM requires many data points to be captured per °C transition with, critically, the highest possible well-to-well precision (i.e. well-to-well thermal and optical uniformity). New software tools are also required for HRM data analysis. The Rotor-Gene 6000 real-time rotary analyzer was engineered specifically for HRM. It incorporates a specially tuned high-intensity optical channel, high-speed data capture (up to 1000 data collection points per °C transition), extreme thermal resolution (0.02°C) and dedicated analysis software. World-best thermal precision (about fifty times better than a traditional block-based system) is one reason the Rotor-Gene 6000 is the industry benchmark for multi-well HRM performance. Click for further information on HRM instrumentation
SNP Genotyping by High Resolution Melt (HRM) Discrimination of human ACTN3 (R577X) SNP genotypes (C to T substitution) using SYTO9 intercalation dye (no probes). Homozygous wild type, mutation and heterozygote samples are shown on a standard normalized melt curve (A) and a difference plot normalized to mutant samples (B). Amplification and HRM analysis was done using a Rotor-Gene 6000 instrument and genotypes were auto- matically assigned by the Rotor-Gene software. The fragment was pre-amplified using a 40-cycle fast protocol (46 min. run time).
Applications HRM has renewed interest in the utility of DNA melting for a wide range of uses, including:
>> Gene Scanning for mutation discovery, fingerprinting
>> Sequence Matching e.g. species/strain typing, HLA compatibility typing
>> Genotyping of of SNPs, haplotypes and repeat sequences
>> Quantitative Allelotyping e.g. prevalence and acquired mutation ratios
>> Quantitative DNA Methylation Analysis e.g. for early cancer detection and monitiring
>> Genetic Linkage Mapping using any marker that generates a melt polymorphism
SYBR and SYTO are registered trademarks of Invitrogen Corporation. EvaGreen is a trademark of Biotium, Inc. LC Green is a trademark of Idaho Technology.
