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Brotec 05-29-2008 02:04 PM

High Resolution Melt
 
Dear all,

I'm interested in the new technique High Resolution Melt (HRM). With this technique I want to analyse the PCR products to look whether they contain mutations/polymorphisms or not.

By melting the PCR product the meltcurve is made by the software by measuring the optical features. The picture of the curve depends on the sequence of the PCR product. When there's a variation in this sequence, the meltcurve will be affected and could be distinguished from the wildtypes.

That looks useful when you want to save money (sequencing the PCR products is expensive). Most samples will not be affected by mutations and they don't need to be analysed by the sequencer. When the meltcurve of 1 PCR product is clearly affected, this sample will be sequenced to take a look what sort of mutation it contains.

So here a question. Do you have some experience with this technique HRM?
And does it help to save money by sequencing less samples? And some tips/help are very welcome.

I'll be very thankful for your help.
Grtz Brotec

danfive 05-29-2008 02:54 PM

Re: High Resolution Melt
 
I'm sure you can find a lot of articles on PCR melting curve analysis for mutation discovery or allele classification. And you can do it with any real-time pcr machine.

Your idea reminds me of the product from this company ---> [Only registered and activated users can see links. Click Here To Register...]

The LightScanner is the first and only instrument to utilize Hi-Res Melting™ for mutation scanning and genotyping in a 96- or 384-well plate system. This exciting technology, licensed exclusively to Idaho Technology, is based upon to the following patent publications: 2005-02333335 (Nucleic and Melting Analysis with Saturation Dyes) and 2006-0019253, (Amplicon Melting Analysis with Saturation dyes).

The LightScanner is unsurpassed in its temperature control and data acquisition rates. With superb sensitivity and specificity, this instrument is designed to meet the needs of high throughput scanning projects. Simply add LCGreen® dye to the initial PCR reaction– then transfer the PCR plate to the LightScanner for melting. Intuitive analysis software groups the results and provides automatic results.

danfive 05-29-2008 03:03 PM

Re: High Resolution Melt
 
Quote:

Originally Posted by Brotec (Post 7942)
Dear all,
I'm interested in the new technique High Resolution Melt (HRM). With this technique I want to analyse the PCR products to look whether they contain mutations/polymorphisms or not.

Good Idea, I've done gene mapping using gels (3-4% agarose most of the time), but melting curves can be as accurate for distinguishing between two differently sized bands. I've also used melting curve analysis for RT-PCR products and I was very satisfied, the melting curves can look really great as long as you have good consistent extraction of DNA/RNA and optimized PCR (no extraneous bands).


Quote:

Originally Posted by Brotec (Post 7942)
So here a question. Do you have some experience with this technique HRM?
And does it help to save money by sequencing less samples? And some tips/help are very welcome.

I'll be very thankful for your help.
Grtz Brotec

Obviously not sequencing the non-mutants will save you money, and you can probably save money by using conventional pcr ingredients with SYBR, you know not paying the premium for a kit.

danfive 05-29-2008 03:16 PM

Re: High Resolution Melt
 
Here are a few pdfs

DNA Melting Analysis: Benefits of High Resolution Systems for Mutation Scanning and Genotyping ---> [Only registered and activated users can see links. Click Here To Register...]

Hi-Res Melting For Mutation Scanning Using LCGreen Plus Dye ---> [Only registered and activated users can see links. Click Here To Register...]

PCR Optimization for Hi-Res Melting Applications --> [Only registered and activated users can see links. Click Here To Register...]

Brotec 05-30-2008 09:13 AM

Re: High Resolution Melt
 
Dear danfive,

Thanks for the links :thumbs:. I've learned much more about this technique :)

Here in my lab there's an appropriate apparatus to perform the HRM. The 7500 fast real-time PCR system of Applied Biosystems. There's a software for HRM available. To purchase this software much money is required... :spcartmanmad:

It would be very desirable to hear somebody have positive experience with comparable apparatus and software to be installed in the existing PC.

Thanks in advance.

nrothe 08-18-2008 06:58 PM

Re: High Resolution Melt
 
Brotec, I am also looking for others using the ABI 7500 or ABI 7900 for mutation detection. I'm currently trying to standardize the protocol using some known SNPs. I have the analysis software, and so far, the results have been inconsistent. Have you found anyone knowledgeable on this topic? any success? Tech support hasn't been responsive......
Best,
Nolan

Brotec 08-28-2008 10:59 AM

Re: High Resolution Melt
 
Dear,

To detect mutation/polymorphism from the PCR product, the product wouldn't be bigger than 250 bp, since at 1 mutation in large amount of nucleotides the curve will be too slight affected you cannot distinguish it from curves of 'healthy' products.

I'm still not started with this technique, since my director is still investigating the possibilities/alternatives.

Maybe there's joined new people in this forum, where somebody are experienced with this technique, I'd be glad to hear experiences with this technique HRM.

Thanx in advance!
Brotec

rameshnrcog 11-11-2008 06:43 AM

Re: High Resolution Melt
 
I would like to know which is the cheap and the best fast PCR machine with which we can do High resolution melt

Brotec 02-03-2009 01:23 PM

Re: High Resolution Melt
 
I don't think there's a cheap means to perform the HRM technique.

It's still not performed here in my hospital, unfortunately. I've been busy with other works...
But last week I started the PC of the 7500 to perform a Taqman assay and I discovered the new software is installed! It happened secretly, I think :D

When the HRM is performed for the first time, I'll write the story of experiences here :)

sparow 04-07-2009 01:18 PM

Re: High Resolution Melt
 
Hi Guys..
Am confused weather to procure Lightcycler 480(Roche) or ABI-7500 fast or RotorGene 6000 (Corbett)..
Kindly considering the running cost, most easy use software and best results as per industry standards..


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