Dual-stage amplified fluorescent DNA sensor based on polymerase-Mediated strand displacement reactions

Abstract

A sensitive fluorescent strategy for DNA detection is developed based on a dual stage amplification process mediated by DNA polymerase. In the sensing method, there are two kinds of DNA probes used, including a hairpin probe (HP) and a double-stranded DNA (DSD) tagged with a quencher and a fluorophore, respectively. The presence of the target DNA triggers the two-step cyclic amplification reactions with the assistance of DNA polymerase, thus leading to the considerable fluorescence restoration. The experiment conditions are carefully studied to achieve optimal performance, including the construction of DSD, the concentration of HP, the reaction time, and the amounts of polymerase. Under the optimal conditions, the strategy responded linearly to DNA target from 0.5 to 50.0 nM with a relative low detection limit of 0.38 nM (according to 3σ/S rule). Moreover, the method shows good detection performance in serum samples, confirming its practical usage for efficient DNA assay. The mix-and-measure simplicity and dual-stage signal amplification make the developed method a novel way for sensitive detection of DNA.