PCR-independent Reagent-Free Nucleic Acid Detection

描述（由申请方提供）：本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，除了与100 nm直径微珠偶联的互补序列捕获探针外，不需要任何特殊试剂。最近的研究表明，纳米孔中单个大分子的存在会导致孔电阻的可测量变化。我们的概念通过放大来自特定单分子靶标的信号以产生容易检测的开/关电流信号，使该单分子检测系统进一步迈出了重要的一步，这是由于孔电阻的非常大的持续增加。所提出的系统的关键要素包括肽核酸（PNA）捕获探针缀合到~100 nm直径的聚苯乙烯球。PNA寡聚体是DNA和RNA的不带电荷的类似物，其共享相同的碱基化学并且与互补NA序列强烈杂交。由于球形-PNA缀合物携带很少或不带电荷，因此它们不表现出响应于跨越含有纳米孔的固态膜施加的稳定DC场的电泳运动。然而，在捕获靶DNA或RNA序列时获得的大量负电荷将使杂交的缀合物在电泳上移动的。如果纳米孔尺寸逐渐变细至小于100 nm的直径，则预期携带杂交的PNA和靶NA的带电缀合物将进入孔的大端并显著增加其电阻，从而引起测量电流的非常强的持续下降。以这种方式，预期该系统给出基本上二元的响应，信号通知靶NA的存在或不存在。这种提出的新技术将是有用的应用，其中确定存在或不存在的NA的特定序列，而不是其浓度，是主要关注的问题，如在患者筛选流行病期间，肿瘤状态评估手术期间，检测食品污染物，和生物战剂检测。 公共卫生相关性：我们提出开发一种用于检测特定核酸序列的装置，该装置基于通过核酸已经结合到其上的珠对纳米孔的堵塞的电测量。该设备产生一个强大的，基本上是二进制的信号，可以很容易地报告与简单的电路，一种方法与传统的PCR和基于荧光的核酸检测技术的主要优势。该技术的潜在应用包括：传染病诊断、遗传性疾病的基因筛查、个性化医疗、犯罪学、亲子关系纠纷、动植物育种、流行病期间的患者筛查、手术期间的肿瘤状态评估、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.