Fluorometric detection of mutant DNA oligonucleotide based on toehold strand displacement-driving target recycling strategy and exonuclease III-assisted suppression.

We describe here a fluorometric assay for sensitive detection of oligonucleotides, based on a target recycling amplification strategy driven by toehold-mediated strand displacement reaction and on exonuclease III (Exo Ш)-assisted fluorescence background suppression strategy. The network consists of a pair of partially complementary DNA hairpins (HP1 and HP2) with 3' overhang ends, between which the spontaneous hybridization is kinetically hindered by the stems. The target DNA is repeatedly used to trigger a recycling progress between the hairpins, generating numerous HP1-HP2 duplex complexes. Exo III was then employed to digest the double strand parts of the residual hairpins and the intermediate products. The fluorescent dye, SYBR Green I, binds to the double-strand DNA products and emits strong fluorescence to achieve sensitive detection of the target DNA with the detection limit of 5.34 pM. Moreover, this proposed strategy showed high discrimination efficiency towards target DNA against mismatched DNA and was successfully applied in the analysis of human serum sample.