PCR-independent Reagent-Free Nucleic Acid Detection

描述（由申请人提供）：该项目的总体目标是证明特异性核酸（NA）序列检测新概念的可行性，该新概念不依赖于聚合酶链反应（PCR）进行靶序列扩增，并且不需要任何特殊试剂，除了与100纳米直径的珠相连的互补序列捕获探针。最近的研究表明，纳米孔中单个大分子的存在会导致孔的电阻发生可测量的变化。我们的概念通过放大来自特定单分子目标的信号，使单分子检测系统向前迈出了重要的一步，从而产生易于检测的开/关电流信号，这是由于孔隙阻力的持续大幅增加。该系统的关键元件包括一个肽核酸（PNA）捕获探针，该探针与直径约100 nm的聚苯乙烯球共轭。PNA低聚物是DNA和RNA的非带电类似物，具有相同的碱基化学性质，并与互补的NA序列强烈杂交。由于球体- pna偶联物携带很少或不携带电荷，因此当施加在含有纳米孔的固态膜上的稳定直流场时，它们不会表现出电泳运动。然而，在捕获目标DNA或RNA序列时获得的大量负电荷会使杂交共轭物在电泳上移动。如果纳米孔尺寸变小到直径小于100 nm，则携带杂化PNA和目标NA的带电共轭物将进入孔的大端，并显著增加其电阻，从而导致测量电流非常强劲、持续的下降。通过这种方式，该系统有望给出一个本质上的二元响应信号，表明目标NA的存在或不存在。这项提议的新技术将有助于确定特定序列的NA是否存在，而不是其浓度，这是主要关注的应用，例如流行病期间的患者筛查，手术期间的肿瘤状况评估，食品污染物检测和生物战剂检测。

DESCRIPTION (provided by applicant): The overall goal of this project is to demonstrate the feasibility of a new concept for specific nucleic acid (NA) sequence detection that does not rely on polymerase chain reaction (PCR) for target sequence amplification and does not require any special reagents other than a complementary sequence capture probe conjugated to 100 nm-diameter beads. Recent work has shown that the presence of single macromolecules in a nanopore causes measurable changes in the pore's electrical resistance. Our concept takes this single-molecule detection system a significant step further by amplifying the signals from specific single molecule targets to generate an easily detected on/off current signal due to a very large sustained increase in pore resistance. The key elements of the proposed system include a peptide nucleic acid (PNA) capture probe conjugated to ~100 nm-diameter polystyrene spheres. PNA oligomers are uncharged analogs to DNA and RNA that share the same base chemistry and hybridize strongly to complementary NA sequences. Since the sphere-PNA conjugates carry little or no charge, they do not exhibit electrophoretic movement in response to a steady, DC field imposed across a solid-state membrane harboring a nanopore. However, the substantial negative charge acquired upon capture of a target DNA or RNA sequence would make the hybridized conjugate electrophoretically mobile. If the nanopore size tapers to a diameter smaller than 100 nm, the charged conjugate carrying the hybridized PNA and target NA would be expected to enter the large end of the pore and significantly increase its resistance, thereby causing a very strong, sustained drop in measured current. In such a way, this system is expected to give an essentially binary response signaling the absence or presence of a target NA. This proposed new technology would be useful for applications where determination of the presence or absence of NA of a particular sequence, rather than its concentration, is of primary concern such as in patient screening during epidemics, oncological status assessment during surgery, detection of food contaminants, and biowarfare agent detection. PUBLIC HEALTH RELEVANCE: We propose to develop a device for the detection of specific nucleic acid sequences based on the electrical measurement of blockage of a nanopore by a bead to which the nucleic acid has bound. The device produces a strong, essentially binary, signal that can be easily reported with simple circuitry, an approach with major advantages over conventional PCR and fluorescent-based nucleic acid detection technologies. Potential applications of this technology include: infectious disease diagnostics, genetic screening for hereditary diseases, personalized medicine, criminology, paternity disputes, animal and plant breeding, patient screening during epidemics, oncological status assessment during surgery, detection of food contaminants, and biowarfare agent detection.