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Old 11-07-2017, 12:50 PM   #1
taylor_wilcox
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Location: USA

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Unhappy Multiplex PCR missing some loci/low template

I am trying to multiplex 12 primer sets in a single PCR. The goal is to amplify 12 taxon-specific loci from environmental samples. So, 1) DNA concentrations are low (<100 DNA copies/rxn) and 2) not all rxns contain all 12 loci (some might contain DNA from only one taxon).

In my no template controls there is a pretty big primer dimer band. I tested all 66 possible primer pairs (n=2 primer pairs per rxn) and found that there is a small-medium size primer dimer band in ~1/2 of them. None of these heterodimers look bad from an in silico test. So I think the big primer dimer band in the full n = 12 multiplex is cumulative. I'm using a lot of cycles (n=40) because template concentration is low.

The problem is that I'm *only* getting a primer dimer band in reactions where template is low (<100 copies/rxn) and there is only DNA from one taxon. I get fewer primer dimers and a better band as I increase template concentration and add in more taxa. I know that in singleplex I get one clean band of the expected size with the same PCR conditions.

My hypothesis: The primer-primer interactions are relatively weak, but without other templates and with lots of cycles, heterodimer formation is taking over the mutliplex PCR.

Does anyone have experience with a similar study design? Having trouble finding the lower limit on template quantities and the effect of missing loci on multiplex PCR. Any ideas on optimization (I know that I could up annealing temp, use TD-PCR, and decrease primer concentrations to start perhaps)?
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Old 11-08-2017, 02:51 AM   #2
avierstr
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I have tried a similar setup. 10 amplicons per multiplex. Only a few amplicons amplified, so I decreased the primer concentration for those amplicons, or increased the concentration for those that not amplified. For some, a missing amplicon appeared, but then an other disappeared. I think I did more than 20 optimization steps in changing concentrations and always some disappear or appear, but I could never get all of them. Also tried different annealing temperatures, MgCl concentrations, number of cycles, all without success.

To conclude, I gave up...
Multiplex sounds fine, but practical is complex to get it running. Maybe with primers with high annealing temperature (above 60-65°) it could work (some kits for amplifying cancer genes work that way), but that was impossible in my experiment.
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