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High resolution melting analysis-SNP genotyping.

assymetric pcr melting curves unlabeled probe SNP genotyping HRMA

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#1 Kaplan.V

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Posted 03 October 2012 - 04:51 AM

Hi ,

I'm trying to genotype one common SNP by High resolution melting analysis.


I'm using short oligos - unlabeled probes, containing single base mismatch.
These probes can succesfully distinguish the mutation (SNP) to homozygous, wild type and heterozygous sample.
( Not much succesfully in my case:(

In short, I prepare the amplicon by assymetric PCR, then I add the probe ( oligo), which hybridizes to the ss DNA ( containing my SNP)
and run it on the LightScanner instrument-this machine is used for high resolution melting analysis- it detects the changes of fluorescence - LC green dye, as the temperature grows up.

Recently I run succesfully a few experiments but now i have problems- no probe hybridization is detected even if I've tried different primers, different primer ratios for assymetric pcr and a various kinds of probes.
My last idea is, that my amplicon is too long-250-300bp and for some reason the place on the ss DNA amplicon could by covered by some kind of hairpin structure.. So I'm getting new primers for amplicon around 100bp. Do you think it can help?

What do you think it's the key to succes in this method?

Does anyone here have some expierences, ideas?

Thanks lot,

V.K.

#2 Trof

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Posted 03 October 2012 - 06:11 AM

AFAIK what you do is not HRM.
You do melting analysis with probes.
On LightScanner you should be able to do classic HRM, (just melting the amplicon itself). The ampliconstrands will bind during strand replication, so you should definitely get some signal. What SNP class do you have? (what kind of substitution)
I don't have experience with this particular melting technique, but shorter amplicons usually help. You can also try some secondary-structure relaxing agents (like DMSO) to add to your reaction if you suspect that. But since you do assymetric PCR are you sure your PCR is running fine? Did you try to evaluate the primers in nonasymetric reactions?

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