Interesting question!
I don't think that it's related to the stability of the polymerase. I also encountered this phenomenon for different poylmerases and kits and supplies and master-mixes. Modern kits contain inactive but heat-activated polymerase that gets activated more and more during the denaturation phases. The inactivated enzymes are quite stable. Further is the concentration of the enzyme so high that even after 50 cycles there will be more than enough enzyme to amplify products to higher concentrations.
In an assay employing hydrolysis probes non-specific amplification products are not detected. The chance that such products occur increases with decreasing concentration of the specific target. The (unobserved but running) amplification of the unspecific products can occupy the polymerase. This may be in part responsible for the differences in the plateau phases. You may check the PCR product on a hi-res gel (6-10% PA) with a highly sensitive staining to get an impression about the specificity of the amplification.
However, I also observe this when using dsDNS binding dyes (SYBR Green) that detect any accumulating product. So there may be other reasons, and I also wonder what these reasons can be.
Interesting question!
I don't think that it's related to the stability of the polymerase. I also encountered this phenomenon for different poylmerases and kits and supplies and master-mixes. Modern kits contain inactive but heat-activated polymerase that gets activated more and more during the denaturation phases. The inactivated enzymes are quite stable. Further is the concentration of the enzyme so high that even after 50 cycles there will be more than enough enzyme to amplify products to higher concentrations.
In an assay employing hydrolysis probes non-specific amplification products are not detected. The chance that such products occur increases with decreasing concentration of the specific target. The (unobserved but running) amplification of the unspecific products can occupy the polymerase. This may be in part responsible for the differences in the plateau phases. You may check the PCR product on a hi-res gel (6-10% PA) with a highly sensitive staining to get an impression about the specificity of the amplification.
However, I also observe this when using dsDNS binding dyes (SYBR Green) that detect any accumulating product. So there may be other reasons, and I also wonder what these reasons can be.
You don't say what dye you are using. With some batches of sybr dyes you may find that with time and heat you are getting a loss of fluorescnce. During each cycle the dye is subjected to excitation either by laser or a halogen light source. Over a number of cycles you may be seeing a "photobleaching" effect - you can see a similar event when doing fluorescence microscopy.
Dear Ian,
I don't think that this is the most probable explanation for that phenomenon. If this would be the case, you would also see a drop of fluorescence after reaching the fluorescence maximum for samples with high template/target concentration.
I myself have seen that happen with different PCR systems. In some cases it might have been kind of competition effect from multiplexing but in other cases it remained unexplainable. I will follow that discussion :).
Best, Stephan
Stephan you may be right. What I have observed on my machine is that there is a point beyond which the machine does not register any increase in fluorescence (100 % saturation, which is determined by the gain settings). Any increase in fluorescence beyond this point is shown as a straight line. Any photobleaching effect is not seen unless the fluorescence falls below the 100% threshold. By increasing the gain settings the visible plateau effect at lower concentrations can be "made to go",(i.e it is now 100% and all saturation appears as a straight line), although the phenomena is still there.
As has been pointed out the point of inflection or Ct value is what really matters not the max fluorescence.
Dear Hans-Joerg,
Interesting approach... Nevertheless, this does not fully explain the problem. Let's assume there is a (total) maximum number of copies that can be generated during the reaction than even a template starting difference of 10e+6 times would not explain the difference in fluorescence plateau. Total numbers of copies generated during PCR (regardless whether the reaction starts with 1 or 1,000,000 copies) is so high that the starting number should not have an effect on plateau max (that would be in the very low percent or per mille range).
There are PCRs that reach a high fluorescence signal even with low starting amounts of target which speaks against this hypothesis.
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