Centre of Reproductive Medicine/Family Planning Research Institute, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
Department of Otolaryngology-Head and Neck Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China.
Mikrochim Acta. 2018 Aug 13;185(9):413. doi: 10.1007/s00604-018-2941-0.
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 (T790 M and L858R) were experimentally demonstrated. The limit of quantification of the MT concentration is 2 nM, and the detection limit of the low abundance of the target sequence is 5% (40 nM of MT strand in the background of 760 nM of WT strand). The fluorometric assay with excitation/emission wavelengths of 485/582 nm 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 2 nM was adopted to multiplexed detection of genetic variations via strand displacement including branch migration and toehold exchange.
一种星形荧光 DNA 探针（S-探针）被描述为能够通过包括分支迁移和引发链置换的链置换来识别靶 DNA 并将其与干扰 DNA 区分开。人工设计的 S-探针在允许荧光标记链和淬灭剂标记链在针对不同遗传变异的不同 S-探针之间共享的同时，不会损害链置换。通常，不同 MT/WT 对的多重检测需要不同的荧光标记和淬灭剂标记链。S-探针的两个标记寡核苷酸与靶/干扰 DNA 序列解耦，因此可以将相同的荧光和淬灭剂链用于针对不同序列的不同 S-探针。该方法对 BRCA 41293497 突变、KRAS G13D 突变和两种类型的 EGFR 突变（T790M 和 L858R）的灵敏度、特异性和通用性进行了实验验证。MT 浓度的定量限为 2 nM，靶序列低丰度的检测限为 5%（WT 链背景下 760 nM 的 MT 链为 40 nM）。采用激发/发射波长为 485/582 nm 的荧光测定法成功地应用于掺入突变型和野生型 DNA 的临床样本。S-探针的独特结构为调节链置换反应提供了有用的工具。可以想象，星形 DNA 探针可以广泛用于遗传变异的多重检测，并为基于 DNA 的纳米机器中利用的链置换过程的调节提供新的见解。
