A star-shaped DNA probe based onstrand displacement for universal and multiplexed fluorometric detection of genetic variations.

Abstract

A star-shaped fluorescent DNA probe (S-probe) is described that can recognize target DNA and discriminate it from interfering DNA via strand displacement including branch migration and toehold exchange. The artificially designed S-probe does not harm the strand displacement while it allows the fluorescently labelled strand and the quencher-labelled strand to be shared among different S-probes targeting different genetic variations. Generally, multiplexed detection of different MT/WT pairs requires different fluorophore-labelled and quencher-labelled strands. The two labelled oligonucleotides of S-probe have sequences decoupled from the target/interfering DNA sequence, so the same fluorescent and quencher strands can be used for different S-probes that target different sequences. The sensitivity, specificity, and general applicability of the method toward BRCA 41293497 mutation, KRAS G13D mutation and two types of EGFR mutations (T790M and L858R) were experimentally demonstrated. The limit of quantification of the MT concentration is 2nM, and the detection limit of the low abundance of the target sequence is 5% (40nM of MT strand in the background of 760nM of WT strand). The fluorometric assay with excitation/emission wavelengths of 485/582nm was successfully applied to clinical samples spiked with mutant-type and wild-type DNA. The unique structure of the S-probe provides a useful tool for the regulation of the strand displacement reaction. Conceivably, the star-shaped DNA probe can be widely adopted to multiplexed detection of genetic variations and provide novel insights into the regulation of strand displacement processes as utilized in DNA based nanomachines. Graphical abstract A star-shaped fluorescent DNA probe (S-probe) with a detection limit of 2nM was adopted to multiplexed detection of genetic variations via strand displacement including branch migration and toehold exchange.