Cтруктура продуктов классической ПЦР и причины выхода реакции на плато

Abstract

Classical PCR with two primers usually produces one amplicon. However, despite the apparent simplicity of this reaction, additional products of different lengths and structures are inevitably synthesized, even when ideal primers are used. These minor products are not given enough attention, but in some cases they can significantly affect the efficiency of PCR, and also contribute to an earlier plateau of the reaction, due to several reasons. The main one is a decrease in the efficiency of primer annealing on the chains of the target amplicon due to an increase in its quantity during PCR and, therefore, an increase in the probability of reassociation of its chains. To describe the processes occurring during PCR, it is proposed to operate with a conventional reaction cell with a volume of 1 zeptoliter (1000 nm3), containing quaternary complexes “DNA/primer/DNA polymerase/dNTP”. The number of ingredient molecules required for PCR to occur per such reaction cell was calculated.