The idea of lost polymerase activity is historical. In the early days of PCR, polymerase enzymes did loose activity with numerous cycles of PCR. Modern thermostable engineered polymerases, however, are quite robust and exhibit nearly full activity at the end of a typical PCR. The idea of one or more of the NTPs or primers being limiting reagents is perfectly logical and consistent with chemical principles but is not correct for the concentrations that are usually used in PCR. Chemical analysis of the PCR mixture reveals that at the end of PCR there is usually plenty of primers and NTPs so that PCR should continue for further cycles before saturation is observed due to consumption of a limiting reagent. Experimentally, if you double the concentration of the primers, you do not observe twice the PCR product. Thus, the amplicon yield of PCR is usually less than predicted based on the primer concentrations.
What is causing the PCR to saturate prematurely? The answer is that double-stranded DNA is an excellent inhibitor of DNA polymerase. This can be demonstrated experimentally by adding a large quantity of non-extensible “decoy” duplex DNA to a PCR and comparing the result to a PCR without the added duplex. The result clearly shows that the reaction with added duplex DNA shows little or no amplification while the control amplifies normally. The reason why duplex DNA inhibits DNA polymerase is that the polymerase binds to the duplex rather than binding to the small quantity of duplex arising from the primers binding to target strands during the early cycles of PCR.
Application of the Inhibition Principle to Multiplex PCR Design