Conventional methods for detecting small quantities of nucleic acids require amplification by the polymerase chain reaction (PCR), which necessitates prior purification and introduces copying errors. While amplification-free methods do not have these shortcomings, they are generally orders of magnitude less sensitive and specific than PCR-based methods. In this review, we provide a practical guide to a novel amplification-free method, single-molecule recognition through equilibrium Poisson sampling (SiMREPS), that provides both single-molecule sensitivity and single-base selectivity by monitoring the repetitive interactions of fluorescent probes to immobilized targets. We demonstrate how this kinetic fingerprinting filters out background arising from the inevitable nonspecific binding of probes, yielding virtually zero background signal. As practical applications of this digital detection methodology, we present the quantification of microRNA miR-16 and the detection of the mutation EGFR L858R with an apparent single-base discrimination factor of over 3 million.

中文翻译：

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单分子动力学指纹图谱核酸检测指南

检测少量核酸的常规方法需要通过聚合酶链反应 (PCR) 进行扩增，这需要事先纯化并引入复制错误。虽然无扩增方法没有这些缺点，但它们的灵敏度和特异性通常比基于 PCR 的方法低几个数量级。在这篇综述中，我们提供了一种新的无扩增方法的实用指南，通过平衡泊松采样 (SiMREPS) 进行单分子识别，它通过监测荧光探针的重复相互作用来提供单分子灵敏度和单碱基选择性。固定目标。我们展示了这种动力学指纹识别如何过滤掉不可避免的探针非特异性结合，从而产生几乎为零的背景信号。