Greetings everyone,
I am aware that in TaqMan probe qPCR's, the annealing and elongation steps are combined into a single step that generally occurs at 60C. I'd like to know if there are any limitations or disadvantages to having a separate annealing step at a lower primer annealing temperature?
I am interested in using a primer set that anneals at 48C. The product is 160 bp long. The probe is being designed, is aimed to anneal at 58-60C to allow it to bind before the primers anneal and dictate the region for copying. I know that this primer set is designed for standard qPCR (absence or presence) and not for probe-based qPCR but what's to stop it from having the standard 3 cycling steps at the cost of a little extra cycling time?
The cycling profile would look something like this (amended to the Soli BioDyne 5x HOT FIREPol® Probe Universal qPCR Mix cycling conditions):
Denaturation: 20s @ 95C
Annealing: 20s @ 48C
Extention: 1 min @ 60C
In theory, this should work. Has anyone had any experience with reactions like this?
When you use the TaqMan probe there is only a initial denaturation (for example 95°C for 5 min) and 24-45 cycles at 2 steps (95°C for 15-30 sec and 58°C-70°C, annealing temperature, for 15-60 sec). This last step depending on the lenght of PCR product and/or number of amplications in tube.
The conditions depend of the producer of master mix. If you have other question
Greetings and thank you for your feedback. You made an interesting point about the annealing/elongation step. So correct me if I read this wrong, the Taq can operate from 58 - 70C but that is dependent on the annealing temp of the Primers and probe.
If possible could you recommend any papers where this has been done. I will greatly appreciate it!
Thank you
Danvir Ramesar you find this information inside the manual of the TaqMan mix that you decide to use. For example I performed a multiplex and I used the TaqMan probe and the thermoprofile is 95°C for 5 min; 40 cycles of 95°C for 15 sec and 60°C for 4 min. In my case the PCR product it is arounf 400-500 bp.
You need to test the probe by qPCR and after check in gel in you have the correct PCR-product. This procedure need time and more test, especially for the validation of the probe.
Which TaqMAn mix did you use?
Hi,
In qPCR, generally the amplicon size will be around 150bp or less. Taqpol can add 100s of nucleotides per second at its optimal temparature ~70- 72degC. Below this temparature Taq will be less active but still be able to add nucleotides.
In combined annealing extension taq adds nucleotides during the ramp up phase from the annealing phase to the denaturation phase.
It's ideal to set primer annealing at 60deg or higher as this will increase the specificity.
If your targeting lower primer annealing due to limitation in probe Tm, you can try MGB or bhqplus to increase the probe Tm.
You can refer the below paper and other papers from the same authors:
Influence of PCR reagents on DNA polymerase extension rates measured on real-time PCR instruments
Jesse L Montgomery et al. Clin Chem. 2014 Feb.
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