I have two targets A and B both are known sequences cloned into plasmids,
The primers Tm for A is 54 and respective probe Tm is 63
The primers Tm for B is 59 and respective probe Tm is 69.
PCR conditions 95-5 min, 40 cycles of 95-30 and 60-30 (detection)
GC contents
Hi,
that sounds quite strange. Maybe you can order a primer with the probe sequence to check if the oligo binds to the target under these conditions resulting in a PCR product. Of course this would not exclude any synthesis error in the sequence of the TaqMan probes. Additional you may change the polymerase and check if the problem is that the probe is not degraded but only displaced by the polymerase. Finally you can check the molecular weight of the probes by MALDI if you have access or you order the same probe again to exclude errors.
Good luck,
Norman
Hello Norman,
I didnt check with another polymerase, reason is the same FAM BHQ1 probe for another target worked absolutely fine with the taq polymerase.
Yes your idea of getting the probe sequence as primer should be considered to rule out any mis match in the target sequence, But both the target sequences were retrieved from NCBI and a blast result of probe sequence did show 100% coverage with the target probe binding site.
I might be expecting a resynthesis of probe from company soon.
and ya considering MALDI TOF, i dnt know if my supplier would be looking for it rather he would resynthesis and give.
But are there any methods to measure the probe and quencher dissociation at 95degree .? I actually dnt know how manufacturers test it after conjugating probe and quencher.? Any idea.
Hello Goutham,
what do you mean with "dissociation at 95°C"? Normally I would expect that the TaqMan probe is quite stable even at 95°C. Longer incubation at this temperature may lead to spontanous hydrolysis, but this should occur for a small fraction of the probe, only. Otherwise the principle of TaqMan qPCR would not work...
Hello Norman
Dissociation of interaction between probe and quencher I mean, it would happen at 95 degrees right.? allowing the probe to become single stranded and at annealing step it gets annealed to target..! Am i right.?
And to add one more test, we did ran the same set of primers and probes even using Solis Biodyne universal probe mix and takara probe mix, no signal at all and yes amplification in gel.
Hi Goutham,
in principle there is no dissociation (no increase of fluorescence) at 95°C for TaqMan probes. It is the case for another qPCR probe format the molecular beacons, where a secondary structure in the probe and the contact between dye and quencher causes quenching of the fluorophore.
Of course a TaqMan probe can show secondary structures which will be resolved before annealing, but this will not interfere with signal generation. Only degraded probes (by the polymerase) will result in increased fluorescence.
Hello Norman
Apart from it what could be the other reasons for the probe not to get annealed to the target, I did check with primer blast using F` with R` and probe with R` both did gave specific hits for the target region.
Do you think the primer and probe Tm parameters and distance are optimum to work in a real time PCR.?
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