Abstract：
Single nucleotide polymorphisms (SNPs) are closely associated with many biological processes, including genetic disease, tumorigenesis, and drug metabolism. Accurate and efficient SNP determination has been proved pivotal in pharmacogenomics and diagnostics. Herein, a universal and high-fidelity genotyping platform is established based on the dual toeholds regulated Cas12a sensing methodology. Different from the conventional single stranded or double stranded activation mode, the dual toeholds regulated mode overcomes protospacer adjacent motif (PAM) limitation via cascade toehold mediated strand displacement reaction, which is highly universal and ultra-specific. To enhance the sensitivity for biological samples analysis, a modified isothermal recombinant polymerase amplification (RPA) strategy is developed via utilizing deoxythymidine substituted primer and uracil-DNA glycosylase (UDG) treatment, designated as RPA-UDG. The dsDNA products containing single stranded toehold domain generated in the RPA-UDG allow further incorporation with dual toeholds regulated Cas12a platform for high-fidelity human sample genotyping. We discriminate all the single-nucleotide polymorphisms of ApoE gene at rs429358 and rs7412 loci with human buccal swab samples with 100% accuracy. Furthermore, we engineer visual readout of genotyping results by exploiting commercial lateral flow strips, which opens new possibilities for field deployable implementation.
摘要：
单核苷酸多态性(SNPs)与许多生物过程密切相关。包括遗传疾病,肿瘤发生，和药物代谢。准确有效的SNP测定已被证明在药物基因组学和诊断中至关重要。在这里，基于双支点调控的Cas12a传感方法，建立了一个通用的高保真基因分型平台。不同于传统的单链或双链激活模式，双支点调节模式通过级联支点介导的链置换反应克服了原型间隔区相邻基序（PAM）的限制，这是高度普遍和超特定的。为了提高生物样品分析的灵敏度，通过使用脱氧胸苷取代的引物和尿嘧啶-DNA糖基化酶（UDG）处理，开发了一种改良的等温重组聚合酶扩增（RPA）策略，指定为RPA-UDG。在RPA-UDG中产生的含有单链立足点结构域的dsDNA产物允许进一步掺入双立足点调节的Cas12a平台,用于高保真人样品基因分型。我们用人类颊拭子样本区分rs429358和rs7412位点的ApoE基因的所有单核苷酸多态性，准确率为100％。此外，我们通过利用商业横向流动条设计基因分型结果的视觉读出，这为现场可部署的实施开辟了新的可能性。
