A kit to detect and count individual DNA molecules of interest

用于检测和计数感兴趣的单个 DNA 分子的试剂盒

• 批准号: 8446255
• 负责人: Glenn Fu
• 金额: $ 15万
• 依托单位: CELLULAR RESEARCH, INC.
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-03 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8446255
• 关键词: Achievement; Area; Bacteria; Biochemical Reaction; Biological Assay; Calibration; Cells; Chemicals; Clinical; Clinical Research; Code; Collaborations; Communicable Diseases; Complex; DNA; DNA Sequence; DNA amplification; DNA analysis; Detection; Devices; Diagnostic; Engineering; Gene Expression; Genes; Genome; Goals; Health; Human; Individual; Label; Malignant Neoplasms; Measurement; Measures; Methods; Molecular Biology; Nature; Nucleic Acid Amplification Tests; Nucleic Acids; Oligonucleotides; Performance; Phase; Process; Public Health; RNA analysis; Reaction; Reagent; Relative (related person); Research; Research Proposals; Sampling; Scientist; Solutions; System; Techniques; Technology; Testing; Time; Tube; Universities; Virus; Work; analog; clinical application; commercialization; comparative; design; detector; digital; disease diagnosis; instrument; interest; member; novel; novel strategies; nucleic acid quantitation; prenatal; prototype; public health relevance; research facility; research study; single molecule

DESCRIPTION (provided by applicant): Accurate measurement of nucleic acid concentrations is essential in many areas of genome research and its application to human health. Sensitive, precise and reliable quantitative nucleic acid tests are increasingly necessary in many important public health problems, including cancer, infectious disease and prenatal diagnostics. However, there is currently a lack of practical technologies capable of high precision measurements, creating an unmet need in research and clinical applications where very small differences in concentration must be discerned. We have recently developed a high-sensitivity counting method with the statistical power of digital PCR, yet can be multiplexed to measure many genes simultaneously. In this proposal, we will develop this technology into a single molecule detection system which will allow absolute counting of single copies of nucleic acids. For phase I of this proposal, our objective is to develop a prototype assay kit and detector to make precise measurements of gene expression. Although many techniques in modern molecular biology allow for the relative quantitation of nucleic acids, digital PCR is the only method capable of absolute quantitation. However, it requires expensive reagents and instruments, and is limited to measurements of a single target at a time. In contrast, our novel method first randomly labels every copy of a DNA molecule with an oligonucleotide barcode tag. After PCR amplification, a small detector panel of complementary oligonucleotides is constructed to detect the number of different barcode tags, which reveals the number of original molecules in solution. This transforms the difficult task of counting individual molecules of identical DNAs into the simple process of detecting the number of different amplified barcoding sequence tags present. Because our method expands identical molecules into chemical space, we can perform the measurement in a single tube, making it much simpler and amenable to multiplex target detection than digital PCR where a very large number of separate containers is required. Control nucleic acids of known concentrations will be tested, and independent measurements will be conducted with digital PCR in order to validate the technique. Our company is comprised of an exceptionally strong team of successful innovators and scientists/engineers with significant achievements in both research and product commercialization settings. Additionally, we have established collaborations with scientists at the Stanford University Genome Technology Center, giving us access to instruments and expertise available at this world class research facility.

描述(由申请人提供)：准确测量核酸浓度在基因组研究及其对人类健康的应用的许多领域是必不可少的。在许多重要的公共卫生问题上，包括癌症、传染病和产前诊断，敏感、准确和可靠的定量核酸测试越来越必要。然而，目前缺乏能够进行高精度测量的实用技术，这在研究和临床应用中产生了一个未得到满足的需求，在这些应用中，必须辨别出非常微小的浓度差异。 我们最近开发了一种高灵敏度的计数方法，具有数字聚合酶链式反应的统计能力，但可以多重同时测量多个基因。在这项提案中，我们将把这项技术发展成单分子检测系统，使之能够对核酸的单拷贝进行绝对计数。对于这项建议的第一阶段，我们的目标是开发一种原型分析试剂盒和检测器，以对基因表达进行精确测量。 虽然现代分子生物学中的许多技术允许对核酸进行相对定量，但数字聚合酶链式反应是唯一能够进行绝对定量的方法。然而，它需要昂贵的试剂和仪器，而且一次只能测量一个目标。相比之下，我们的新方法首先用寡核苷酸条形码标签随机标记DNA分子的每个副本。聚合酶链式反应扩增后，构建一个由互补寡核苷酸组成的小探测器板来检测不同条形码标签的数量，从而揭示溶液中原始分子的数量。这将计数相同DNA的单个分子的困难任务转变为检测存在的不同条形码序列标签的数量的简单过程。由于我们的方法将相同的分子扩展到化学空间，我们可以在单个试管中进行测量，使其比数字PCR更简单、更易于进行多目标检测，因为数字PCR需要非常大量的单独容器。将对已知浓度的对照核酸进行测试，并将使用数字聚合酶链式反应进行独立测量，以验证该技术。 我们公司由一支由成功的创新者和科学家/工程师组成的特别强大的团队组成，他们在研究和产品商业化方面都取得了重大成就。此外，我们还与斯坦福大学基因组技术中心的科学家建立了合作关系，使我们能够使用这一世界级研究设施提供的仪器和专业知识。

项目成果

Digital encoding of cellular mRNAs enabling precise and absolute gene expression measurement by single-molecule counting.

• DOI: 10.1021/ac500459p
• 发表时间: 2014-03-18
• 期刊: ANALYTICAL CHEMISTRY
• 影响因子: 7.4
• 作者: Fu, Glenn K.;Wilhelmy, Julie;Stern, David;Fan, H. Christina;Fodor, Stephen P. A.
• 通讯作者: Fodor, Stephen P. A.