I am trying to amplify two DNA targets in a duplex PCR for diagnostic purposes.
Targets A and B are normally amplified in singleplex PCR with their respective primers. Targets A and B are also normally amplified in duplex PCR (both primers) when the sample is only one of the targets. But target A is not amplified in duplex PCR when adding samples to both A and B, only B is amplified. This has been confirmed in qPCR and electrophoresis. Why is this happening? And how to solve it?
Sample concentration is very similar between A and B. I also tried to increase A's primer concentration in the duplex mix and no changes were seen.
Adding more primer dna,target dna and ntps can reduce the amount of free MG ions and the polymerase requires at least 1mM Mg to be an active enzyme. I wonder if the addition of extra primer is reducing the free Mg in the reaction. Try running mixed samples wit 1, 2, 3 and 4 mM Mg concentrations. this Mg gradient will also address any annealing problems caused by insufficient Mg ( annealing temps are lower in higher Mg concentrations and reducing the free Mg in your reaction may raise the Ta of one primer set so that one primer set works poorly
I know it sounds like an odd idea, but have you tried increasing the reaction volume? Sometimes things that don't amplify in a 25 microliter reaction will work in a 50 microliter reaction.
Also, how large are the amplicons from each target? You need at least a 10% size difference to tell molecules apart on an agarose gel.
What happens if you have Target A and Target B with just A primers? How about Target A and Target B with just B primers?
Hi Katie A S Burnette Great questions!
We did test with 50uL reaction and the results were the same.
Their size is somewhat similar, so to be sure we used qPCR (where the mix with both primers and targets showed only B TM) and also tested using labeled primers detected by antibodies.
And yes we tested that too, A primers with A and B samples produce only A amplicon (with slight 2CT delay). B primers with A and B samples produce only B amplicon without problems.
Hi Paul Rutland Amazing suggestion! I hadn't thought about that. I'll be doing it as soon as possible.
Hi,
When multiplexing with qPCR, the amplification of one target can interfere with the amplification of the other target. B might be preferentially amplified and push back the amplification of target A.
A few things to consider:
- design of your primers: be sure there are no primer dimers etc. and melting temperatures are similar for the primers and the probes (for the probes a bit higher than for the primers) of both targets
- amplicons need to have a similar size (shorter = better, 60-150bp ideally). Longer amplicons will amplify less efficiently.
- if one target is more abundant than the other (so ~ if Cq-value B
Hello Caroline Weydert ! Thank you for your feedback, I will keep these in mind.
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