TaqMan PCR assay
If my primrs are not specific for that species, can I still use very specific probe (2-3bp difference) to get the required specificity.I designed my primers according to standrd guidlines and when checking on the sequnce it is different(has 1-2bp difference), but when I check my primers on normal PCR it amplifies all the species. My targeted gene is IGS.Please help me to get species specificiy in my TaqMan assay.? Do you have any sugessions to improve my experimets.Thanks alot
Hello,
if you perform an endpoint PCR and your primers are not really specific for a single species but for many species than you will get a band on your agarose gel. In contrast to endpoint PCR the signals you get with your TaqMan assay are based on the binding of the probe. So the signal you get with your probe might still be species specific. Have you thought about adding a melting curve analysis after your TaqMan amplification? If you have a positive control for the species you want to amplify you could define a melt point for it. This would add more specificity to your assay.
Best wishes
Dirk
Hi
Just to add something to Dirk's comment:
If you want to check the specificity of the probe by melt curve analysis, (by Tm comparison as Dirk suggested), you'll have to use it as a primer and in a new assay using a binding dye such as Sybrgreen.
You won't be able to run a melt curve with a Taqman assay.
David
Hi David and Dirk,
Thanks very much for your reply. As you suggested I think is best way to order my probe as a just primer and check for its specificity.I will also use it with SYBER green assay. I haven't ordered the TaqMan probe yet, as I am hesitate do that as my primers are not specific to my targeted species yet.
1) So according to your idea, even if the primers are not specific, if the probe is very specific can still detect the targeted species?
The primers are not really necessary to be species specific???
2) One of my designed primers has some specificity, however forward primer has very low GC content (26%). Reverse (47%) .But the probe in between has 55% GC content. Primers are working. but bit faint band in the gel. Do you think I can use that in TaqMan assay?
3) I heard from Applied Biosysytems about MGB TaqMan probe.Do you have any idea about that? will it add more specificity for my assay?
4) Also I read on papers about SNP rich primers/probes? As they are polymorphic regions, if I designed my primers targeting those areas will it add any advantage than designing primers using a software (Primer3, primer BLAST). I read in a paper, they used one SNP area as the probe in TaqMna assay and it worked.
5) As I worked with very closely related species (Plant pathogen -Colleototrichum acutatum complex), the IGS sequences are very similar to each other.It is bit hard to detect unique regions within that.Do you have any suggestion to improve the primer/probe designing process?
I am sorry..!I know its lot of questions.I have no qPCR experience so far and I am still learning.This is a part of my PhD project. If you could share some of your expertise ideas on this really appreciate. And it will help most of the others as well.
Thanks..!
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