In my opinion the reason for a single annealing/extension step is simply to save time. Most of the modern DNA polymerases used in the qPCR master mixes have sufficient activity at 60C (20-30bases/sec).
Therefore is you follow the general guides to keep the amplicon as short as possible (80-120bp), using a separate annealing and extension step will not make a big difference in term of Cq and efficiency.
In fact, although most the qPCR guides recommend 1min annealing/extension step, 30 sec are more than enough if you have a short amplicon! The added advantage is that you gain specificity and save time.
As there are always exceptions, I would use a three-step protocol when the target is "difficult" - when you can't design primers with Tm higher than 55C. Obviously in that case the extension will be very slow, so I'd give it a push by adding another step at 72C.
Is a very interesting question and difficult to obtain the answer.
I suppose that the reason is a consequence of the using of TaqMan probes. When you use a TaqMan probe, this needs to be hybridized to your template (annealing temperature) and at the same time the Taq must to be amplifying, and the dsDNA-specific 5'-3 exonuclease activity cleaves the reporter. So the Taq must be working at the same time that the probe must be hybridized.
So I suppose that in the aims of generate a universal protocol, independent of the probes that you were using (SYBR-Green of TaqMan probes), they designed the protocol in this way.
I'm not sure of this, is just an idea.
Good luck!
thank you for your answer and it make sense, but it is not suggested only for TaqMan probes but even for specific primers using SYBr green detection with Tm about 60 °C.
qPCR primers are USUALLY distinguished by higher Tm than those used for cloning/genotyping/other stuff. Since the polymerase has broader temp. activity than the 72C, AND you want to reduce the uncontrolled amplification (REALTIME!!!), hence minimizing such events is crucial.
Intriguing question: Thank you for making me think.
In my opinion the reason for a single annealing/extension step is simply to save time. Most of the modern DNA polymerases used in the qPCR master mixes have sufficient activity at 60C (20-30bases/sec).
Therefore is you follow the general guides to keep the amplicon as short as possible (80-120bp), using a separate annealing and extension step will not make a big difference in term of Cq and efficiency.
In fact, although most the qPCR guides recommend 1min annealing/extension step, 30 sec are more than enough if you have a short amplicon! The added advantage is that you gain specificity and save time.
As there are always exceptions, I would use a three-step protocol when the target is "difficult" - when you can't design primers with Tm higher than 55C. Obviously in that case the extension will be very slow, so I'd give it a push by adding another step at 72C.
thank you for your detailed answer and it is satisfactory. Usually I keep separate the annealing temperature (30 sec) and extension 30 (sec) with amplicon from 80-250 bp. Do you think that there are differences if you perform or not primers concentration adjustment in the two systems?
I think primer concentration adjustment should always be performed before any qPCR measurement/ new amplicon. Although I can't exactly remember the title now, I remember I read an article that examines if asymmetrical primer concentration optimization ( for an example Forward 100 450 900 nM vs Reverse 100 450 900 nM) worth the efforts and I remember that only in about 15% of all amplicons studies it was advantageous to use different concentration of F and R.
Most qPCR guides recommend final primer concentration of 250nM and this is what I also found optimal for my SYBR-based assays.
Having all other conditions constant between a two-step or three-step protocol, the primer concentration could be increased in the reaction that has lower efficiency and higher Cq . To check your efficiency, make a pool of cDNA from all samples, make serial dilutions and check the slope of the curve Cq vs. [cDNA].
thank Petar, I will try to find the paper that your read, it's sound interesting. Thanks for you comments!
Though it is true in most TaqMan that you can get good results using 60 TA without extension, I recently adjust one of my test's TA from 60 to 56 and found significant improvement on sensitivity.
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