Blocking uncertain mispriming errors of PCR

The polymerase chain reaction (PCR) plays a central role in genetic engineering and is routinely used in various applications, from biological and medical research to the diagnosis of viral infections. PCR is an extremely sensitive method for detecting target DNA sequences, but it is substantially error prone. In particular, the mishybridization of primers to contaminating sequences can result in false positives for virus tests. The blocker method, also called the clamping method, has been developed to suppress mishybridization errors. However, its application is limited by the requirement that the contaminating template sequence be known in advance. Here, we demonstrate that a mixture of multiple blocker sequences effectively suppresses the amplification of contaminating sequences even in the presence of uncertainty. The blocking effect was characterized by a simple model validated by experiments. Furthermore, the modeling allowed us to minimize the errors by optimizing the blocker concentrations. The results highlighted an inherent robustness of the blocker method in that fine-tuning the blocker concentrations is not necessary. Our method extends the applicability of PCR and other hybridization-based techniques, including genome editing, RNA interference, and DNA nanotechnology, by improving their fidelity.