![]() Things become slightly more complicated than this because organisms contain two ( or more) copies of each gene, known as the two alleles. 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. So now the two melt curves appear different. However, if one person has a mutation in the DNA region you have amplified, then this will alter the temperature at which the DNA strands melt apart. If you are comparing two samples from two different people, they should give exactly the same shaped melt curve. It is dependent on the sequence of the DNA bases. The melting temperature of the amplicon at which the two DNA strands come apart is entirely predictable. The machine then simply plots this data as a graph known as a melt curve, showing the level of fluorescence vs the temperature: The HRM machine has a camera that watches this process by measuring the fluorescence. But as the sample is heated up and the two strands of the DNA melt apart, presence of double stranded DNA decreases and thus fluorescence is reduced. In the absence of double stranded DNA they have nothing to bind to and they only fluoresce at a low level.Īt the beginning of the HRM analysis there is a high level of fluorescence in the sample because of the billions of copies of the amplicon. They bind specifically to double-stranded DNA and when they are bound they fluoresce brightly. The dyes that are used for HRM are known as intercalating dyes and have a unique property. This is achieved by using a fluorescent dye. The key to HRM is to monitor this separation of strands in real-time. At some point during this process, the melting temperature of the amplicon is reached and the two strands of DNA separate or "melt" apart. The process is simply a precise warming of the amplicon DNA from around 50 ˚C up to around 95 ˚C. This region that is amplified is known as the amplicon.Īfter the PCR process the HRM analysis begins. In the sample tube there are now many copies of the DNA region of interest. 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. HRM analysis is performed on double stranded DNA samples. 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. It is fast and powerful thus able to accurately genotype many samples rapidly.This makes it ideal for large scale genotyping projects. other genotyping technologies such as sequencing and TaqMan SNP typing. It has advantages over other genotyping technologies, namely: It was discovered and developed by Idaho Technology and the University of Utah. 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. ( Learn how and when to remove this template message) ![]() JSTOR ( October 2011) ( Learn how and when to remove this template message).Unsourced material may be challenged and removed.įind sources: "High-resolution melting analysis" – news Please help improve this article by adding citations to reliable sources. This article needs additional citations for verification.
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