PCR-independent Reagent-Free Nucleic Acid Detection

不依赖 PCR 的无试剂核酸检测

• 批准号: 8095463
• 负责人: Harold George Monbouquette
• 金额: $ 20.11万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-20 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8095463
• 关键词: Amines; Animals; Base Sequence; Binding; Biological Warfare; Breeding; Caliber; Charge; Chemistry; Communicable Diseases; Criminology; DNA; DNA Sequence; Detection; Devices; Diagnostic; Discrimination; Disputes; Drops; Electrical Resistance; Electrolytes; Elements; Epidemic; Exhibits; Food; Genetic Polymorphism; Genetic Screening; Goals; Hereditary Disease; Ionic Strengths; Measurable; Measures; Medicine; Membrane; Methods; Movement; Nucleic Acid Probes; Nucleic Acids; Nucleic acid sequencing; Nucleotides; Operative Surgical Procedures; Oral cavity; Patients; Peptide Nucleic Acids; Plants; Polymerase Chain Reaction; Polystyrenes; RNA; RNA Sequences; Reagent; Reporting; Resistance; Sampling; Scheme; Screening procedure; Signal Transduction; Silicon Dioxide; Single-Stranded DNA; Solutions; Surface; System; Tars; Technology; Testing; Work; analog; base; electric field; electrical measurement; father role; macromolecule; nanometer; nanopore; new technology; nucleic acid detection; response; sensor; single molecule; solid state

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.

描述（由申请人提供）：该项目的总体目标是证明新概念对特定核酸（NA）序列检测的可行性，该序列检测不依赖于聚合酶链反应（PCR）进行目标序列扩增，并且不需要任何特殊的试剂，并且除了互补序列捕获概率以外的其他特殊试剂捕获对100 Nm-diame-Diame-diame-Diame-diamet-diamet-diamet-diamet-diamet-diamet-diamet-diamet-diamet-diamet-diameter beads。最近的工作表明，纳米孔中的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特定单分子靶标的信号来使该单分子检测系统进一步取得重要的一步，以生成易于检测到的ON/OFF电流信号，这是由于孔电阻的持续持续增加而引起的。所提出的系统的关键要素包括肽核酸（PNA）捕获结合到〜100 nm的聚苯乙烯球体的探针。 PNA低聚物是对DNA和RNA的无负荷类似物，它们具有相同的碱基化学，并强烈杂交与互补NA序列。由于球体-PNA偶联物几乎没有电荷，因此它们不会表现出电泳运动，因为响应构成纳米孔的固态膜上施加的稳定的DC场。但是，捕获靶DNA或RNA序列后获得的大量负电荷会使杂交共轭物在电泳上移动。如果纳米孔尺寸的锥度至小于100 nm的直径，则预计带有杂交PNA和靶NA的带电共轭物将进入孔的大端，并显着提高其电阻，从而导致非常强大的，持续的测量电流下降。以这种方式，预期该系统将给出基本的二元响应，以表明目标NA的存在或存在。这项提出的新技术对于确定特定序列的存在或不存在而不是集中的NA的应用将很有用，例如在流行病学期间患者筛查，手术期间的肿瘤学状态评估，食物污染物的检测和生物脂肪剂的​​检测。 公共卫生相关性：我们建议开发一种基于核酸与核酸结合的珠子对纳米孔阻塞的电气测量的特定核酸序列的装置。该设备产生一个强，本质上是二元信号，可以通过简单的电路轻松报告，这种方法比常规PCR和基于荧光的核酸检测技术具有主要优势。该技术的潜在应用包括：传染病诊断，遗传性疾病的遗传筛查，个性化医学，犯罪学，亲子症纠纷，动物和植物育种，流行病期间的患者筛查，手术期间的肿瘤学状态评估，食物污染物的检测以及生物脂肪剂的​​检测。