I am trying to determine the usefulness of the marker shown in the attached photo. You will see that in one sample there appears to be 3 alleles (this is a diploid, or more precisely a dikayotic spore). I have seen this in other samples too and have eliminated the possibility that it is DNA contamination by looking at other markers that don't show this pattern.
I have already gone through the step of Pig-tailed reverse primer (these are the results for this) and a longer final extension in the PCR (30 minutes) to try to eliminate stutter on the 'rooster comb' peaks you see. Any other suggestion, or just don't use this marker?
If you have other markers that give better results just drop this one. It could take a long time to troubleshoot and optimize.
If you do want to troubleshoot, try increasing the annealing temp, adding some magnesium, or lowering your dNTPs.
If you really are curious as to what all is amplifying you could sequence your products. But, that information might not be truly helpful for optimizing this marker.
What organism do you work in? Often times large genomes (or ones not well-characterized) will have some unexpected sequence similarity.
Hi Vick, I wouldn't worry about that if I was you. Amy gave you some interesting tips and you can go ahead if you want to, but I think this one you are seeing is just a technical artifact. Some florescence reading chambers, as those from ABI3500, are too sentitive to florescence signals in fragment runs and some times very strong signals extrapolate the notation of one length wave to another. Therefore, we usually see a reading from, let's say NED- fluorescence at the exactly same location in the FAM or HEX (standard ABI chemicals).
If you are loking at a multiple samples view, they seem real peaks, but if you see that at multiple flourecence view (all length waves from the same individuals) you will probably find out that your individual has coincident peaks in multiple florescences at the same time. It should be hugely high at the original flourecence but smaller than the actual ones at the others.
If you did a single-flourescence run, it is also possible to be a neighbor signal noise noted to another sample.
I've seeing that a hundred of times with different samples and different organisms and repeating PCR with different flourecence or getting less florescence at the run make the strange peaks gone. You might also test it by diluting your pcr product a bit more before the run.
Try to check this out before doing more tests. If you still getting such a peak then you can think about other possibilities. If you have any questions about specificities, feel free to message me.
Hope it might be helpful.
Best
Amy and Wilson,
Thank you for your excellent answers. Amy, I am working with the fungus Puccinia graminis (stem rust). My DNA is from urediniospores, which contain two haploid dikaryons, so I view them somewhat like diploids with two alleles per sample.
Wilson, I did check for pull-up peaks and don't see that, but I do think that it is quite possibly due to too highly concentrated neighboring samples. Difficult to get concentration correct in multiplex samples to be able to read all dyes. That poor PET dye is so faint, but with work I am getting this better.
Still, I have other markers I may substitute just to make it easier, but this was well worth learning the finesse of multiplex capillary electrophoresis.
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