Apart from the apparent exhaustion of the dNTPS during the PCR cycles there are several factors that come into play that could play a significant role in determining the amount of final product. Although the exhaustion of dNTPs or primers may come into play but it is generally such that they have a higher concentration than necessary. so couldn't be the only reason. Another scenario would be the case of enzyme denaturation, upon constant exposure to the continuous cycles of amplification, there would come a time when the efficiency of the enzyme is gradually lost. However, when the cycles are kept constant for the different amount of template concentration, though there would definitely be change in the amount of final product but one cannot exceed beyond certain concentration, now this may be due to the presence of excess amount of the template itself. In other words, in a case where you start with lesser amount of template, the log phase of the cycle could continue for a long time before reaching the plateau effect; Similarly, where you start with the high amount of template the log phase would be lesser but the amount of product would be higher, although the plateau effect would eventually be reached. Hence, what I am trying to convey is that, the inhibition due to template concentration would eventually come out to limit the amount PCR amplicons, as in due to crowding effect, no matter the concentration of template, there is only so much that it can contribute to the overall concentration of the product at the end. In some extreme cases, the higher amount of template could even inhibit the entire PCR amplification resulting in no amplification at all.

Furthermore, taking absorbance reading of the PCR products could give false report, as the underutilized primers as well as dNTPs could also absorb the wavelength. Hence, to sum up, it could be that the concentration of the template itself be the limiting factor, in a sense that, higher template concentration results in PCR inhibition.

Hope this is helpful.

Apart from the apparent exhaustion of the dNTPS during the PCR cycles there are several factors that come into play that could play a significant role in determining the amount of final product. Although the exhaustion of dNTPs or primers may come into play but it is generally such that they have a higher concentration than necessary. so couldn't be the only reason. Another scenario would be the case of enzyme denaturation, upon constant exposure to the continuous cycles of amplification, there would come a time when the efficiency of the enzyme is gradually lost. However, when the cycles are kept constant for the different amount of template concentration, though there would definitely be change in the amount of final product but one cannot exceed beyond certain concentration, now this may be due to the presence of excess amount of the template itself. In other words, in a case where you start with lesser amount of template, the log phase of the cycle could continue for a long time before reaching the plateau effect; Similarly, where you start with the high amount of template the log phase would be lesser but the amount of product would be higher, although the plateau effect would eventually be reached. Hence, what I am trying to convey is that, the inhibition due to template concentration would eventually come out to limit the amount PCR amplicons, as in due to crowding effect, no matter the concentration of template, there is only so much that it can contribute to the overall concentration of the product at the end. In some extreme cases, the higher amount of template could even inhibit the entire PCR amplification resulting in no amplification at all.

Furthermore, taking absorbance reading of the PCR products could give false report, as the underutilized primers as well as dNTPs could also absorb the wavelength. Hence, to sum up, it could be that the concentration of the template itself be the limiting factor, in a sense that, higher template concentration results in PCR inhibition.

Hope this is helpful.

Likely you are correct that dNTP exhaustion is the limiting factor.

Quick calculation using typical PCR reaction conditions:

200uM dNTPs, ~490 g/mol molecular weight for dNTPs

200*10^-6 mol/L * 490 g/mol = 0.098 g/L dNTP's in rxn mix (~100mg/L)

100 mg/L --> 100 ng/uL dNTPs

I was just curious how estimated the concentrations of your PCR product. Was it Nano Drop based or fluorescence based?

Secondly, did you purify before estimating the concentrations?

Answers to the above could also help in deciphering the above question.

Other than that @Mitesh has very summarized the answer to your query.

PCR has many rate limiting factors, and adding more template does not equal to more product. Infact excess of template would give you lower product. I think if you just need higher concentration of your product, run multiple PCRs and combine them, I do this routinely and I get good amount of DNA. Another thing you could try is scaling up your reaction, i.e. increasing the reaction volume.

Hope this helps!

You can also use the purified PCR product as the template for further rounds of amplification.

I agree with Mitesh Shrestha comment above.

Template inhibition is due to competence between template and primers. Likewise, the "plateau effect" is primarily due to increased primer-template competence as template concentration increases along PCR cycles.

As mentioned above, using more template will not help your situation. Why not optimize your anealing conditions. Do a gradient PCR. also check the age of your polymerase and make sure its still very active.

Just to point out, excessively high number of cycles may give you non-specific binding as you may already know.

finally check to make sure your buffers have the right amount of salts.

Good luck with your experiments.

Thanks everyone for the insightful answers

agree with @ Mitesh Shrestha

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Hi Joseph,

I know it is late in coming but it is an interesting question so thought I would add my opinion.

Most of the answers here are correct there are a lot of limiting factors, however I feel the limitation to concentration of DNA lies with the primers.

Chris gives a nice calculation with the DNTPs, but the calculation is based upon a 200 um concentration, when it would be 200um of each DNTP. However this does not mean there will be 4 times as much product as when incoperated into the growing DNA chain the mw of a DNTP will fall closer to 330 daltons sor more like a limit of around 300ng/ul.

However the general primer concnetrations in PCR are around 1 uM or 1 pmole/ul. If you imagine that it runs at 100% efficiency that would be 1 pmole/ul of product per ul.

in the case of a 500bp product that is about 330 ng/ul of DNA (1pmol *330000 daltons) (I am taking 660 daltons as the average weight of a base pair).

However you will start running out of primer far below this, as you need two primers on the same template to amplify a specific product. Also you need the primers in excess to compete against the complementary strand trying to reanneal to get binding of the template, so primers are probably the limiting factor for small products, whereas you may start running out of DNTP's for larger products (as there is more DNA for same number of primers)

There is also the fact that conditions in the tube becoume unfavourable at later cycles, denaturation, pH changes, DNTP availability, ionic changes etc.

Howver the two most limiting factors are primers and DNTP's it is these that you would need to increase to increase concentration. However This leads to other problems such as mageasium depletion by DNTPs and the fact that a lot of the amplification will be carried out in unfavourable conditions at the end. So easiest thing is to increase reaction size and concentrate after, which also gets rid of most of the unwanted bits from the reaction.

I am sure you are aware of that, but you did pose an interesting question that made me want to figure it out