PCR-independent Reagent-Free Nucleic Acid Detection

描述(由申请人提供)：本项目的总体目标是证明一种新的特定核酸(NA)序列检测概念的可行性，该概念不依赖于聚合酶链式反应(PCR)进行目标序列扩增，并且除了连接到100 nm直径的珠子上的互补序列捕获探针外，不需要任何特殊的试剂。最近的研究表明，纳米孔中单个大分子的存在会导致该孔的电阻发生可测量的变化。我们的概念将这种单分子检测系统向前推进了一大步，通过放大来自特定单分子目标的信号，由于孔阻持续大幅增加，从而生成易于检测的开/关电流信号。该体系的关键元件包括与直径约100 nm的聚苯乙烯小球连接的肽核酸(PNA)捕获探针。PNA寡聚体是DNA和RNA的不带电荷的类似物，它们具有相同的碱基化学，并与互补的NA序列强烈杂交。由于球形-PNA偶联物携带的电荷很少或没有电荷，因此它们不会随着施加在含有纳米孔的固态膜上的稳定的直流场而表现出电泳运动。然而，在捕获目标DNA或RNA序列时获得的大量负电荷将使杂交的偶联物具有电泳性。如果纳米孔尺寸逐渐变小，直径小于100 nm，则携带杂化PNA和目标NA的带电共轭有望进入孔的大端，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，该系统预计会给出一个本质上是二元响应的信号，表示目标NA的存在或缺失。这项拟议的新技术将适用于以下应用：确定特定序列的NA的存在或不存在，而不是其浓度，例如流行病期间的患者筛查、手术期间的肿瘤状态评估、食品污染物的检测和生物制剂的检测。 与公共卫生相关：我们建议开发一种设备，用于检测特定的核酸序列，该设备基于对与核酸结合的珠子阻塞纳米孔的电子测量。该设备产生的信号很强，本质上是二元的，可以用简单的电路很容易地报告出来，这是一种比传统的PCR和基于荧光的核酸检测技术具有重大优势的方法。这项技术的潜在应用包括：传染病诊断、遗传疾病的基因筛查、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、食品污染物的检测和生物制剂检测。

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.