High Resolution Melt (HRM) analysis is a powerful technique in molecular biology for the detection of mutations, polymorphisms and epigenetic differences in double-stranded DNA samples.
Typically the user will use polymerase chain reaction (PCR) prior to HRM analysis to amplify the DNA region in which their mutation of interest lies.
The difference may only be tiny, perhaps a fraction of a degree, but because the HRM machine has the ability to monitor this process in "high resolution", it is possible to accurately document these changes and therefore identify if a mutation is present or not.
Conventional SNP typing methods are typically time-consuming and expensive, requiring several probe based assays to be multiplexed together or the use of DNA microarrays.
The HRM method has been successfully used to detect a single G to A substitution in the gene Vssc (Voltage Sensitive Sodium Channel) which confers resistance to the acaricide permethrin in Scabies mite.
The analysis of scabies mites collected from suspected permethrin susceptible and tolerant populations by HRM showed distinct melting profiles.
The amplicons from the sensitive mites were observed to have a higher melting temperature relative to the tolerant mites, as expected from the higher thermostability of the GC base pair[3] In a field more relevant to clinical diagnostics, HRM has been shown to be suitable in principle for the detection of mutations in the breast cancer susceptibility genes BRCA1 and BRCA2.
HRM offers a faster and more convenient closed-tube method of assessing the presence of mutations and gives a result which can be further investigated if it is of interest.
In a study carried out by Scott et al. in 2006,[4] 3 cell lines harbouring different BRCA mutations were used to assess the HRM methodology.
Similarly in 2007 Krypuy et al.[5] showed that the careful design of HRM assays (with regards to primer placement) could be successfully employed to detect mutations in the TP53 gene, which encodes the tumour suppressor protein p53 in clinical samples of breast and ovarian cancer.
The consensus is that HRM is a cost efficient method that can be employed as an initial screen for samples suspected of harbouring polymorphisms or mutations.
These methods include the use of PCR with specifically designed probes to detect the variants of the genes (SNP typing is the simplest case).
A study by Gundry et al. 2003,[6] showed that fluorescent labelling of one primer (in the pair) has been shown to be favourable over using an intercalating dye such as SYBR green I.
[8] Another study compared the predictive power of MGMT promoter methylation in 83 high grade glioma patients obtained by either MSP, pyrosequencing, and HRM.
HRM also is practically advantageous for use in diagnostics, due to its capacity to be adapted to high throughput screening testing, and again it minimises the possibility of amplicon spread and contamination within a laboratory, owing to its closed-tube format.
[12] For accurate and repeatable results, PCR thermal cycling conditions must be optimized to ensure that the desired DNA region is amplified with high specificity and minimal bias between sequence variants.
For this reason, it is critical to accurately predict the melting curve of PCR products when designing primers that will distinguish sequence variants.