Multiplex PCR does not work - (Nov/07/2011 )
I have a problem with my multiplex PCR.
I created a reaction with 10 PCR-Markers. 2 are FAM-labelled, 2 are Ned-labelled, 3 are Pet-labelled, 3 are VIC-labelled.
When I tried them all alone just within their dyes, the reaction was OK. But when I put all the 10 Markers together, the reaction does not work. I think some Markers are interacting with each other, but I don't know which ones . Is there a programm or something like this in the internet, where I can give my sequences to and perhaps find out which markers do not fit/are interacting with each other?
Thank you very much,
I don't know if I understand you well, did you put all 10 markers in a single tube?? If you did that then thats the problem. The multiplex fuction when you have 2 or 3 probes labeled different if they aren't in the same spectral measure. There is no way that the machine can discriminate between 2 FAM or 3 of any of the same label. For example you can make a multiplex with 1 FAM and 1 VIC and you should not have problems because FAM exitation max is 494nm and emission 518nm for VIC is 538 and 552 respectively. For NED is 546nm & 575nm so it is a little bit near VIC so that could cause problems. About PET I didn't find the information of the spectrum, but if it is near any of the others can't be use in a multiplex. When doing a multiplex you have to optimize the primer/probe concentration because you will have several of them but the same concentration of enzyme and nucleotides and they will compete.
But if the sizes of the amplicons with the same dye are different enough, it can work. But not all primer might work together but will form primer dimers etc. Also the annealing temperatures should be similar enough.
Multiplx is an online software to find out primers that (might) work together, anyway you have to do a lot of optimising as merlav already mentioned. Usually it's trial and error and the more primer pairs you have the more difficult it gets, and some primers just don't work together and you never find out why.
Yes, I put all the 10 Markers in 1 Tube. This works in theory, because I have reactions with 4 VICs in it or 3 Pets and this works. Because the fragments are far away from each other.
As you have said, I think I'll have to change the problematic markers into an other Multiplex (we have 115 markers in 12 reactions...). Perhaps I will never find out why some markers don't work. I used the internet site : http://eu.idtdna.com/analyzer/Applications/OligoAnalyzer/
and I found out that 2 markers make hetero-dimers with 11 base pairs. That's very bad, isn't it?
Thank you very much for the help, I'll try the Multiplx-link also!