Abstract
AbstractClinically and biologically, it is essential to detect rare DNA-sequence variants for early cancer diagnosis or drug-resistance mutations identification. Some of the common quantitative PCR (qPCR)-based variant detection methods are restricted in the limit of detection (LoD) because the DNA polymerases used have a high polymerase misincorporation rate, thus the detection sensitivity is sometimes unsatisfactory. With the proofreading activity, high-fidelity (HiFi) DNA polymerases have a 50- to 250-fold higher fidelity. However, there are currently no proper probe-based designs as the fluorescence indicator allowing multiplexed HiFi qPCR reactions, thus restricting the application of HiFi DNA polymerases like the variant detection. We presented the Occlusion System, composed of a 5’-overhanged primer with fluorophore modification and a probe with a short-stem hairpin and a 3’ quencher modification. We demonstrated that the Occlusion System allowed multiplexing HiFi qPCR reaction, and it was compatible with the current variant-enrichment method to improve the LoD up to 10-fold. Thus, the Occlusion System satisfactorily functioned as efficient fluorescence indicator in HiFi qPCR reactions and allowed application of HiFi DNA polymerases in variant detection methods to improve detection sensitivity.
Publisher
Cold Spring Harbor Laboratory