Tuning the specificity of DNA probes using bulge-loops for low-abundance SNV detection
Tuning the specificity of DNA probes using bulge-loops for low-abundance SNV detection
复制标题
使用凸环调整 DNA 探针的特异性以进行低丰度 SNV 检测。
DOI：
10.1016/j.bios.2020.112092
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发表时间：
2020-04-15
影响因子：
12.6
通讯作者：
Xie, Guoming
中科院分区：
文献类型：
--
作者：
Bai, Shulian;Xu, Bangtian;Xie, Guoming
调节分子探针与靶标之间的自由能差被认为是实现选择性突变识别的可行方法。但是由于探测序列的变化程度有限，因此简单地通过改变探针的碱基组成来适度地利用热力学动力学仍然是一个挑战。在此，我们提出了调制的单核苷酸变异（SNVs）检测的歧视能力插入到双链体DNA探针的凸出环。基于在与突变或野生型DNA的链交换之前和之后自由能变化（Δ G）的可控调谐，获得比常规线性探针高得多的特异性。所提出的凸出环探针允许在富含鸟嘌呤和胞嘧啶（GC）的区域中优异地区分SNV，并且在低至2飞摩尔的靶基因下达到0.02%丰度的检测限。该探针在鉴定肺组织样品中的低丰度L858 R突变体时也表现出与液滴数字PCR（ddPCR）的优异一致性，所述低丰度L858 R突变体不能通过商业PCR试剂盒或桑格测序来分辨。我们的工作不仅为床旁诊断链交换探针的合理设计提供了见解，而且还更广泛地推进了动态DNA纳米技术中可定制级联反应的构建。
Tuning the free energy difference between a molecular probe and the target has been regarded as a feasible way to realize selective mutant recognition. But due to limited extent of variation on the probing sequences, it remains a challenge to moderately leverage the thermodynamic kinetics simply by changing the base composition of probes. Herein we propose the modulation of discrimination capability for single nucleotide variations (SNVs) detection by insertion of bulge-loop into duplex DNA probes. Based on controllable tuning of free energy change (Delta G) before and after strand exchange with either mutated or wild-type DNAs, much higher specificity than conventional linear probes is obtained. As-proposed bulge-loop probes allows excellent discrimination of SNVs in high guanine and cytosine (GC) rich regions, and reaches a detection limit of 0.02% abundance with down to 2 femtomolar target gene. The probes also demonstrate excellent consistence with droplet digital PCR (ddPCR) in identifying low abundant L858R mutant in lung tissue samples that are not resolved by either a commercial PCR kit or Sanger sequencing. Our work not only provides insight into the rational design of strand exchange probes for point-of-care diagnosis but also advance the construction of customizable cascade reactions in dynamic DNA nanotechnology more broadly.