Hairpin structure facilitates high-fidelity DNA amplification reactions in both qPCR and high-throughput sequencing

Effective polymerase chain reactions (PCR) are important in bio-laboratories. It is essential to detect rare DNA-sequence variants for early cancer diagnosis or for drug-resistance mutations identification. Some of the common detection quantitative PCR (qPCR) methods are restricted in the limit of detection (LoD) because of the high polymerase misincorporation rate in Taq DNA polymerases. High-fidelity (HiFi) DNA polymerases have a 50- to 250-fold higher fidelity. Yet, there are currently no proper designs for multiplexed HiFi qPCR reactions. Moreover, the popularity of targeting highly multiplex DNA sequences requires minimizing PCR side products, as the potential of dimerization grows quadratically as the plexes of primers increases. Efforts tried before were either an add-on step, or technology-specific, or requiring high-level computing skills. There lacks an easy-to-apply and cost-effective method for dimerization reduction. Here, we presented the Occlusion System, composed of a 5'-overhanged primer and a probe with a short-stem hairpin. We demonstrated that it allowed multiplexing high-fidelity qPCR reaction, it was also compatible with the current variant-enrichment method to improve the LoD by 10-fold. Further, we found that the Occlusion System reduced the dimerization up to 10-fold in highly multiplexed PCR. Thus, the Occlusion System satisfactorily improved both qPCR sensitivity and PCR efficiency.