Entering edit mode
5.6 years ago
dom.byrne13
▴
20
Hi,
Essentially I need a program which can estimate whether or not a qPCR reaction would fail or not given a set of primer/probe sequences and the target sequence which would be being amplified. For the wider application I need this estimate to be relatively realistic, so I was wondering if anyone knew of any tools which could be used at the command line to do this sort of thing? If not, any advice on how to construct a set of weights for various mismatch types/positions to attempt to do it myself would be very much appreciated.
Have you looked at any of the tools available for designing TaqMan primers? I would expect they can do this.
It's a complicated problem, and will depend on the environmental context: I'm interested in a tool which can tell me if a qPCR amplification will "go well" when the sample is a blood extraction from Model Organism X. We may have to define "go well" and specify the amplification parameters.
typically, PCR success is mostly seen as being dependent on the interaction strength of the target sequence and the primer and its specificity. Of course, DNA degradation will play a role in how well preserved the full stretch of your target sequence is, but I doubt this can easily be modeled (although there might be some insights into typical break points of DNA). In short, I concur with Devon in thinking that there should be numerous tools out there doing a pretty good job at helping you design PCR primers. Have you looked through the list provided by MolBioTools?
This might also be a RT-qPCR question, in which case also "is the targeted gene/transcript sufficiently expressed" becomes important.
So due to the nature of my specific problem, I'll be testing whether established PCR primers will amplify different substrates. So the problem is markedly simpler in that I don't have to worry about too many of the issues associated with primer design (e.g. self-annealing, Tm etc.) and my question comes solely down to the number/type/position of mismatches (I think!). Unfortunately this seems to rule out many of the primer design tools I've looked at, unless I'm being particularly dense (which is not to be ruled out).