Hi all
I'm working with qPCR and I persistently find 2 peaks in my qPCR analysis. While my PCR target is 127bps, I see a peak at 82°C and a sharp peak at 88°C. I checked it by agarose electrophoresis and PAGE and Interestingly I've got one single band in both experiments. I'd like to see if anybody knows the reason that I see one sigle band while I have 2 sharp peaks in qPCR?
Thank you in advance
I've seen this happen for 2 reasons:
1. You have primer dimers. These typically melt at a really low temp & will make a discrete low temp peak. Easy to tell on an agarose gel. Hard to get rid of & still have good primer efficiency.
2. If your PCR product has regions with very different GC% content, then a single DNA molecule will melt in phases. The lower GC% region will melt first, which causes a partial drop in fluorescence. Then the higher GC% melts and causes the second drop in fluorescence. You'll get a "shouldered" or double peak. Either decide it's OK to have that sort of data or you have to design new pairs of primers.
Good luck!
Seconding Katie A S Burnette 's point 2, here, especially since most primer dimers melt at 70-75.
I have an amplicon that is about 50% GC overall, but 80% of that is in one half of the sequence, and it melts in exactly this sort of fashion (80 and 86). Conveniently for me, a SNP I'm interested in also sits just at the midpoint, and affects the melt peak of the lower (but not the upper) band, so I can use my melt curve diagnostically: 80/86 WT, 78/86 mutant.
The best option would be to re design primers to prevent the above issue. Also sequence the product and check for the right product or any variation, especially SNP.
Thank you all dear coworkers, your answers really helped me.
John Hildyard Vanita Malekar
https://www.qiagen.com/us/resources/faq?id=fa36da79-0ffc-42c9-af19-822e7a7d9529&lang=en
It may help you
Sima Ravaei
I agree with Katie A S Burnette . I used to apply this free software (https://www.dna-utah.org/umelt/quartz/um.php) to analyze in silico melt curves. Please, put your amplicon in the box, select the conditions and be amazed. It is pretty easy and good to predict what Katie A Burnette described before.
Also, read this https://www.idtdna.com/pages/education/decoded/article/interpreting-melt-curves-an-indicator-not-a-diagnosis
Best regards,
Jacó
Thank you Jacó Joaquim Mattos very much.
I used the link you shared and it showed one single peak in the melting curve!
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