Your resolution to melt

Posted by John on 28 June 2010 | 0 Comments

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Amplify them all and let the melting sort ‘em out.
OK perhaps my attitude to analysing sequence variation may be a little flippant but it characterises a lot of my thoughts toward High Resolution Melting Analysis (HRMA).

It is now more than a decade since melting analysis to characterise PCR products was introduced. This technique, typically after SYBR Green-based real-time qPCR, is an economic and flexible mainstay of research laboratories around the world for applications such as gene expression due to their ease of design and reduced cost (just primers, no labeled probe(s) required).

In recent years, the advancements in dyes for qPCR, instrument heating precision and new software algorithms are now doing the same for sequence variation analysis – meaning high resolution melting analysis  is becoming widely-established as a rapid, cost-effective and far-ranging means for rapid sequence analysis.

Originally described for SNP variant analysis (and still the leading application), HRMA is now being used in a wider context such as HLA comparisons, microsatellite genotyping and methylation status of DNA sequences. Even within SNP analysis, HRMA may be applied in several ways – scanning regions for heterozygotes (and possibly homozygous variants) or for genotyping a known SNP. Newer developments such as unlabeled probes and snapback elements on PCR primers allow the simultaneous genotyping of a desired SNP along with the scanning of the amplicon for any other sequence variation.

New methods and new applications means then exponential trend of HRMA publications is unlikely to plateau for quite some time. Get melting!

You may wish to view:

‘ HRMA – beyond the SNP’  (webinar on www.bitesizebio.com/seminars). Webinar on 22 June, 2010 - recorded.

Suggested reading

  1. Montgomery JL, Sanford LN, Wittwer CT. High-resolution DNA melting analysis in clinical research and diagnostics. Expert Rev. Mol. Diagn. 2010;10:219-40.
  2. Erali M, Wittwer CT. High resolution melting analysis for gene scanning. Methods 2010;50:250-61.
  3. . . . anything else by CT Wittwer
  4. Vossen RH, Aten E, Roos A, den Dunnen JT. High-resolution melting analysis (HRMA): more than just sequence variant screening. Hum. Mutat. 2009;30:860-6.
    Highly recommended review describing a number of applications for HRMA

If you're doing HRMA already, please add a comment with your application - always great to hear new ideas


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