High primer concentration improves PCR amplification from random pools.

The success of PCR is partly based on its exponential amplification characteristics. Nevertheless, in practice, the achievable yields are limited. Figure 1A and B shows the quantification of a typical reaction with varying but defined amounts of starting template. Every five cycles an aliquot of the reaction was removed and afterwards the amount of accumulated product quantitated. Although, initially, the amplification rates of all reactions reached the theoretical limit of 32-fold amplification during five cycles (indicated by a dashed line), a sharp decline in amplification rates was observed at later cycles. Interestingly this decline, which occurred in all reactions, did not depend on the total number of cycles in each reaction, but rather seemed to be coupled to the amount of accumulated product. In order to learn more about the basis of this phenomenon, I investigated the effect of primer concentration on PCR. For standard applications a primer concentration between 0.1 and 1 μM is recommended (1), and rarely the primers are completely used up during the reaction. Nevertheless, the primers have to compete with the accumulating product in finding their target sequence, which could become limiting for the reaction at late cycles. To test whether the ratio between primer and product concentration influences the product yield, I performed reactions with increasing amounts of primers but otherwise identical conditions. For all reactions tested, an increase in product yield at late but not early rounds of amplification was observed. Nevertheless, the level of improvement strongly