Many reported SYBR green multiplex PCR assays gave few significant peak within the melting curve.
I have designed 2 primer sets for two different target in my project. 500nM primers were used for the 10 uL volume reaction.The size of amplicons are 80 bp and 107 bp respectively. Product of qPCR run gave 2 peak (Tm1= 76, Tm2=80) with 4°C differences. But the size another peak was too small when I tested with different amount of gDNA (please find attached).
From there I postulate the efficiency of each primers were affected by amount of template presence in the reaction tube. What I can in order to improve smaller peak to a more significant size?
I would be grateful to receive any thought from you. Correct me if I am wrong.
Thank you.
Dear Dr. Amin,
Thanks for the response. My question is as above.
I have attached the result I obtained while performed duplex qPCR. Could you briefly explain why the size of the peak is different when low and high template were used.What should I do in order to increase the value of the smaller peak. In other words, how to increase the space occupied by the smaller peak.
I hope you get what I mean =)
Hi,
Multiplex qPCR is certainly doable with SYBR green, even it might not be the ideal tool to do so. The distance between melting peaks is a subject of Tm, so if you further increase the Tm difference, you will get a bigger gap - I assume the two different peaks represent the two different primer pairs.
With that, and that will depend on the type of experiments you plan with this system, you might also want to run standard curves for both primer pairs to compare their efficacy.
Hi again,
"Do you think 5°C is enough to distinguish the two peak? Based on my result as pasted above,"
Most likely yes, as long as you get consistent results (eg the same primer pairs always result in the same melting peak). You can also run the PCR product on gel to confirm that you got the amplicons with the right size.
"In lower gDNA sample assay, second peak primer seemed to be less efficient do you agree?"
Difficult to say for certain from one image; I would certainly run a standard curve with a dilution row, you would then have a much better estimate
Hi Peter,
I am just curious. Theoretically, for dilutions derived from the same source, I supposed peak patterns for low [gDNA] to be lower yet similar as compare to high [gDNA]. However, in my case, the peak patterns for both template showed contradiction. I guess there is a competition in the reaction. Please correct me if I am wrong.
I was thinking to try different combination of primer concentration. Hopefully to get a consistent result with two significant peak in one reaction. And absolutely not forget to generate a standard curve as you suggested. Thanks for the informative thought!
Hi Lee,
Most certainly there is a competition between primers. Just to be sure i understand correctly, the two different peaks on the image (Tms 76 and 80) are the results of two different primer pairs, is that correct? Or peaks shifted due to different amount of Dna but using the same primer pair?
Great, so as long as your no template controls produce different peaks, you got yourself an assay. Run standard curves to see if the two systems can reach the same sensitivity/efficacy - if not, try different annealing temperatures/times, and like you said different primer cc can help as well. Good luck!
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques