High Resolution Melt Analysis

After the DNA Extraction and the ICE COLD-PCR enrichment process, your lab may choose to run the High Resolution Melt Analysis (HRM):
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.
HRM advantages over other genotyping technologies:
  • Cost effective vs. other genotyping technologies such as sequencing and TaqMan SNP typing.
  • Fast and powerful thus able to accurately genotype many samples rapidly.
  • Simple. 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.

Spot the difference

The melting temperature of the amplicon at which the two DNA strands come apart is entirely predictable. It is dependent on the sequence of the DNA bases. If you are comparing two samples from two different people, they should give exactly the same shaped melt curve. 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. So now the two melt curves appear different. 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.