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High Resolution Melting (HRM) Problem - (May/31/2011 )

Dear Colleagues,

Im using High Resolution Melting (HRM) approach (Roche) for the gene screening of a gene with 4 exons. I have got fine results for exons 2-4 but i cant get the result for exon 1. Im doing all the same like i did with other 3 exons but in each run there is a very low amplification and the increase in the fluorescence signal is very late (after cycle 37). That makes impossible to distinguish samples that have mutations since they are recognized by software either as a non template samples or there is too many samples group based on their Tm.

So far, i have tried to optimize reaction with classic PCR troubleshooting (MgCl2, annealing Tm...) and i have also tried another primer for the same exon but the result wasnt any better. I have also made the parallel runs of the same samples with different primer combinations at the same run,and i got result for the other exons but not for this problematic first exon again.

These primers that im using have additional small part of M13 sequence (other colleagues are using them for the sequencing),so i would like to ask if that can somehow affect HRM reaction and should i perhaps use classical PCR primers instead.

Im kinda running out of ideas what could be a problem with this problematic first exon so any advice or idea what could be wrong or if you experienced the similar problem in the past, would be very useful.

Many thanks for your help. :)

-vinx8-

I would try primers without anything added, that's for sure.
Just an idea, isn't your problematic exon GC rich? Because if you have low amplification with different primers, that would point to it.
<Doctor House> Treat with 5% DMSO! Either it will work or patient dies. </Doctor House>

-Trof-

Trof on Tue May 31 12:35:39 2011 said:


I would try primers without anything added, that's for sure.
Just an idea, isn't your problematic exon GC rich? Because if you have low amplification with different primers, that would point to it.
<Doctor House> Treat with 5% DMSO! Either it will work or patient dies. </Doctor House>

That part of the sequence has GC ratio around 55% so thats not a problem. :)
Amplification is good for the other exons.

-vinx8-

The overall CG ratio could hide a region with high GC (but that's not that likely in 55% overal, unless the rest of the exon is AT rich), maybe check it with GC plot to be sure. There could be only single exon (or a part of it)in whole gene that's GC rich, that's usually the case.

You can try DMSO or betaine anyway (or other enhancers, see this for example), sometimes they enhance PCR for unknown reason.

Obvious advice is to design yet another primers, without M13 likely and take good care about their features (primer-dimer formation), what works for classic PCR may not be enough for HRM.

-Trof-

Splitting the exon in two amplicons sometimes helps!

-ElHo-