scorpion probe - (Apr/03/2012 )
Hallo all,
I have a question about the scorpion probe: its works because the probe hybridizes to the DNA that is been formed, but how come the probe hybridizes with this DNA ?
How come the formed DNA doesnt stay together with the template?
It has something to do with intermolecular forces vs intramolecular forces I think, but I dont completely understand this.
Anyway who can explain this?
The probe binds in the next cycle, there is denaturation step before that.
Trof on Tue Apr 3 21:02:17 2012 said:
The probe binds in the next cycle, there is denaturation step before that.
Yes, I know that, but why does the newly formed DNA bind the scorpion probe rather then the complementary template DNA (which is not fully denatured btw, so how come the probe (denatured completely, is able to bind the complementary, made, DNA rather then the template DNA keeps bounded to that made DNA)
I think both things occur, just like binding of a Taqman probe between two DNA strands occurs only in some percentage of cases. The portion of molecules binded is enough to make a visible fluorescent signal.
And I can't be 100% sure of course, but I think denaturation step in each cycle is enough to denature the PCR amplicon, which is usually about 200 bp long.
Trof on Tue Apr 3 21:46:27 2012 said:
I think both things occur, just like binding of a Taqman probe between two DNA strands occurs only in some percentage of cases. The portion of molecules binded is enough to make a visible fluorescent signal.
And I can't be 100% sure of course, but I think denaturation step in each cycle is enough to denature the PCR amplicon, which is usually about 200 bp long.
But I found this: During the next cycle, the hairpin-loop unfolds and the loop-region of the probe hybridizes
(on this webpage: http://www.sigmaaldrich.com/life-science/custom-oligos/dna-probes/product-lines/fluorescent-probes/scorpions-probes.html)
You see, they cleary state its an intramolecular binding... compared to intermolecular normally between 2 complementary strands.
I wonder what the difference is between intermolecular and intramolecular.
That only means that the probe is part of this new strand (because the primer that started it is connected to probe) so binding is "within" the strand. It's on the picture.
Inter- means between, between two different molecules.
Trof on Wed Apr 4 17:22:01 2012 said:
That only means that the probe is part of this new strand (because the primer that started it is connected to probe) so binding is "within" the strand. It's on the picture.
Inter- means between, between two different molecules.
Yes I know what inter and intra means..
But my question is: are those intramolecular forces then stronger then the intermolecular forces and thus "aiding" in the binding of the scorpion probe with the complementary DNA rather then the binding of this complementary DNA with the template DNA.
If I understand you correctly, you mean that the binding of the probe or the template DNA with the complementary DNA happens at random , right? So there is no differences between the intermolecular binding and te intramolecular binding?
Yes, random more or less. The probe has the advantage to be always near the site, but also has to bend the backbone to reach it. But that probably plays a minor role. Since Scorpion probes are said to be more sensitive than Taqman, I would speculate that the vicinity is the important thing. I'm not really sure about which "forces" are you talking.
The strands can be separated during denaturation by far wider distance as I imagine, because even if there are domains that are still aligned those are far apart, like CG rich repeats. And this is only true for the original template, the newly formed amplicons are much shorter and have Tm usually lower than 90, that should be enough to denature completely AFAIK.
Trof on Wed Apr 4 19:40:50 2012 said:
Yes, random more or less. The probe has the advantage to be always near the site, but also has to bend the backbone to reach it. But that probably plays a minor role. Since Scorpion probes are said to be more sensitive than Taqman, I would speculate that the vicinity is the important thing. I'm not really sure about which "forces" are you talking.
The strands can be separated during denaturation by far wider distance as I imagine, because even if there are domains that are still aligned those are far apart, like CG rich repeats. And this is only true for the original template, the newly formed amplicons are much shorter and have Tm usually lower than 90, that should be enough to denature completely AFAIK.
ok.
I have one more question.
I dont know if you checked this website: http://www.premierbiosoft.com/tech_notes/Scorpion.html
If you like at the little animation , it seems that the probe hybredizes to the complementary strand while the complementary strnd and template arent even denatured.. Is this correct? it seems weird to me..
But they also mention this in their explenation: The denaturation of the hairpin loop requires less energy than the new DNA duplex produced. Consequently, the hairpin sequence hybridizes to a part of the newly produced PCR product. This results in the separation of the fluorophore from the quencher and causes emission.
Now I find it pretty weird, the hairpin loops requires less energy to denature then the DNA duplex, but is it possible for the prob DNA to hybredize with the DNA duplex DNA even if that duplex isnt denatured?