High Resolution Melt analysis

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Ivan Delgado
Ivan Delgado's picture
High Resolution Melt analysis

If you have not heard about the latest application of real time PCR, High Resolution Melt (HRM) analysis, read on. This technology makes real time PCR not only much more powerful but also a lot cheaper.
Its power stems from its simplicity. All HRM does is amplify a PCR fragment, just like regular PCR, but instead of ending there it is able to image the melting characteristic of this PCR fragment (with the use of an intercalating dye like SYBR Green). If for example you have PCR fragments of different sequence composition (like when you amplify a DNA region in a heterozygote, which contains both wild type and mutant sequences), these fragments generate melt profiles that are different (G-Cs will melt at a higher temperature than A-Ts, and so on).
Knowing that you can identify any variation in DNA sequence based on its melting characteristic, you can imagine some of the many ways this technology can be used:
1. SNP genotyping: Lets say you have generated a knockout mouse and started backcrossing it to a new background. You could identify an SNP that distinguishes these two lines and design a PCR assay that amplifies this region. Each line would generate a different HRM profile and you could easily keep track of all the mice that contain your insert (by regular PCR or HRM) as well as the new desired background (by HRM). This used to be done using multiplex Taqman assay or arrays.
2. Point mutation detection: lets say are studying how a gene in E. coli mutates over generations when exposed to a given stress. You could design a PCR assay that spans the region you are interested in studying and then start testing the different generations. The HRM melt profile of your first generation will be your baseline and then any mutation that occurs in any future generation will yield an HRM melt profile that would be clearly different from the original. This used to be done by sequencing the whole region every generation.
3. Epigenetics: you still need to perform bi-sulphite treatment to retain the methylation pattern of your DNA of interest, but instead of having to send this DNA for sequencing you can run an HRM assay which will tell you which CpGs are methylated (melting at higher temperatures) and which are not (lower melting temperatures since the non-methylated CpGs are turned into Ts)
And these are just three applications. The ways in which you can use HRM are only limited by your imagination. Think about any situation in your research where you would need to tell DNA sequences apart and there is a good chance HRM will be one of the easiest ways of doing this. Plus it is by far cheaper than the alternatives.
Keep an eye out for HRM, its use in research will grow significantly over the next few years. And if your research justifies it, try to get familiar with it.

R Bishop
R Bishop's picture
Wow that sounds cool.  Is HRM

Wow that sounds cool.  Is HRM technology commercially available yet?  Seems like a logical fall out of staring at all those melting curves after the qPCR run is over.  I can only imagine some scientist in that Eureka moment.
 
Thanks Ivan
 
Rus

Ivan Delgado
Ivan Delgado's picture
Hi Rus,

Hi Rus,
Yes, HRM is commercially available. Pretty much the only two instrument that can do this right now are Qiagen's Rotor Gene Q (previously known as the Rotor Gene 6000 until Qiagen purchased Corbett) and Roche's LC480. Applied Biosystems claims that their ABI 7500 qPCR instrument can perform HRM, but the truth is that it can only do a limited amount of HRM work. This technology is pretty novel so right now obtaining it will cost you (An ABI 7500 costs around $45,000 and the HRM upgrade is another $14,000, although it may have been reduced down to about $3,000 now that you can buy the low end version of the Rotor Gene Q for just under $30,000). I can imagine that within the next year or so HRM technology will be much more affordable.
I can totally imagine a scientist staring at a traditional melt curve for the nth time and thinking: "hey, what if I take a ton more images per second during the melting profile to increase the resolution of my melt profiles?" (that is really what HRM does, with a few other details). This may have been what happened to Carl Wittwer, co-founder of Idaho Technologies, the company that first developed HRM as a qPCR application.
Cheers

CarlosAM
CarlosAM's picture
Hi Ivan. First: do you speak

Hi Ivan. First: do you speak spanish? second: about HRM applications. I would like to  found information about bacterial biodiversity studies using HRM. do you know about, works, proects or groups working in this area?
Thank you. very much
Carlos

Ivan Delgado
Ivan Delgado's picture
 

 
Hi Carlos,
Yes, I speak Spanish (it happens to be my first language). HRM is ideally suited for bacterial biodiversity since you can design an assay that theoretically could identify any variant within the amplicon. Unfortunately I do not know of any group that is working on this area using HRM.
 

marziyeh
marziyeh's picture
dear Ivan

dear Ivan
 thanks for the information that you gave. i would like to know if HRM is used for large DNA base pairs (eg; 1000bp or more)? and is HRM coupled with real-time PCR?

thanx
marziyeh

Ivan Delgado
Ivan Delgado's picture
 

 
Dear marziyeh,

Unfortunately HRM does not work very well with large amplicons. If you go over the literature you find that HRM works best when the amplicons are between 200 and ~600 bp. Once you start getting beyond that size you start getting problems. 

HRM is coupled with real-time PCR in that you need to amplify an amplicon to analyze it my HRM. Having said that, HRM can be performed with any amplicon, even one that was not amplified in the instrument that is performing the HRM.

Cheers