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Melting curve genotyping on LC480


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#1 Trof

Trof

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Excellent

Posted 10 February 2011 - 10:08 AM

I'm writing it here, even when it's not a pure qPCR, but this method uses real-time cycler.

I'm trying to optimize melting curve genotyping on our LightCycler 480. I'm not explaining the method, because if you don't know it you probably can't help me, but basically you have two probes on your SNP and when there is a mismatch the probe melts earlier than if there is different base and the FRET-transfered fluorescence is different. In my case the wt sequence has a mismatch and mutant is matched.

I started with basic recomendation in the GT mix manual from Roche. Melting range from 40 (1 minute incubation) to 75 deg. Now what I got is this:
my-melt-curves.jpg

There are three peaks, the first one same for all samples, the second two are those specific for the SNP, as you can see sample 8 is mutant, sample 5 heterozygous and the rest is wt. You can say you distinguish between the genotypes but it's not very nice.

It should look like this instead (picture on the right):
Posted Image

There are two problems:
  • The first peak shouldn't be there, there seems to be melting something around 45 deg. What is that, free probes maybe? But well, I can try to fix it by starting the melting at higher temperature, like 50.
  • Bigger problem is, that there should be one distinct peak for wt, different one for mutant and both for heterozygote. But my wt samples have two peaks, just lower than heterozygote. Like the probe melts at the right temperature (lower than mutant, because of mismatch), but then it melts also partialy at higher temperature too, the probe seems to sit there for some reason.

There are many things to change (primer/probe concentration, melting range, melting incubation,...) but I'm not familiar or experienced with melting curve GT, so I'm not sure what to change, like would the longer/shorter incubation cause the probe to stop lurking at the wrong temperature?

Has anyone an experience with this type of analysis?
(not a big chance, but still it's better to ask)

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