Dear colleagues!
I recently conducted a qPCR with primers that I've used for many reactions without any problems. I did not change anything, sample material is the same, PCR-dye is the same, water new and clean.
BUT suddenly I observe two peaks in the melt curve. And not constantly. The two pictures attached show two technical replicates pipetted on one 96-well-plate measured in the same reaction. That appeared not as an exception, unfortunatelly I found it in many of my samples. How is this possible to happen?!
By the way, I have not done a gel yet as it would not explain the sudden appearance of the the two peaks...
Thanks a lot in advance for sharing your thoughts!
Primer-Dimers?
Hard to say anything. You'll need a gel picture. And: do the unexpected peaks occur in samples with high and/or with low target concentrations? If the are only in low-concentration samples then this would be a hint towards primer-dimres.
Are these peaks present when you stop the PCR right when the plateau-phase is reached? This would show if this is an "early" or a "late" amplification event. A "late" event is likely unspecific (again pointing towards primer-dimers) and may not neccesarily seriousely interfere with a quantification.
Another possibility: sequence variation, possibly introduced during the PCR. The melting curve can the show the melting of homo- and heteroduplexes. Run 2 subsequent melting curves to check that: 1) right after the last elongation phase do NOT heat and cool, only do the slow temp gradient for the melt curve. If two peaks show up, it is unlikely that it is due a sequence variation (because there should only be homoduplexes present; they may have different Tms but not by several kelvins, they should not be distinguishable in the melt curves). 2) then heat and cool quickly (-> formation of heteoduplexes) and measure the second melt curve. If the peak pattern is changed than it is likely that you have sequence variants. A sequencing may then provide further insights.
Hello Jochen,
thank you for your detailed reply. Primer-dimers was also the first phenomenon I thought of. But I wondered where they might suddenly come from. I mean, as I said, I used these primers for a high number of reactions and I had never Primer dimers or any other kind of problem...
I conducted a gel now, please find a picture attached. Left is a reaction how it should be (product size is 68 bp) and which corresponds to the right melt curve that I sent last time. The middle lane belongs to the left melt curve that I sent last time. I'd like to emphasize again, that both reactions are made of the same master mix, the same program in the same run and even the same sample.
I would appriciate it very much if you could have a look again, thank you!
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