Real-time DNA Sequencing

实时 DNA 测序

• 批准号: 6983963
• 负责人: Susan H Hardin
• 金额: $ 160.22万
• 依托单位: VISIGEN BIOTECHNOLOGIES, INC.
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-01 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6983963
• 关键词: DNA directed DNA polymerase; biomedical automation; biotechnology; computer assisted sequence analysis; fluorescent dye /probe; immobilized enzymes; nucleic acid sequence; technology /technique development

DESCRIPTION (provided by applicant): VisiGen Biotechnologies, Inc. is developing a sequencing system in which polymerase and nucleotides act together as direct molecular sensors of DNA base identity. More specifically, the technology detects the interaction between a modified nucleotide and a fluorescently-modified polymerase. As a nucleotide is incorporated into the growing DNA polymer, energy transfers from an excited donor fluorophore within the polymerase to an acceptor fluorophore within the nucleotide, stimulating the emission of a base-specific incorporation signature that is directly detected in real-time. Cutting-edge technologies, including single molecule detection, fluorescent molecule chemistry, computational biochemistry, and biomolecule engineering, are combined to create this novel sequencing system. DNA samples will be processed in massively parallel arrays that will enable large genomes to be sequenced in less than a day for approximately $1000 and permit sufficient oversampling to produce redundant data that will minimize errors. The system is being developed to identify pathogens (or variations thereof) and to enable comprehensive genome analysis. Proof of principle for VisiGen's technology is demonstrated: sequence information has been detected at the single molecule level in real-time using an immobilized, donor-labeled polymerase and 2 different acceptor-labeled dNTPs. Given the success of our federally-funded efforts, further development of the VisiGen system to sequence arrays of single DNA molecules in real-time is clearly warranted. The specific aims of the proposed research are to refine the sequencing chemistry and to design, build and test the next generation single-molecule DNA sequencing instrument. This instrument, Alpha-1, will be placed at VisiGen and accelerate technology development. This instrument will be used for beta testing by researchers at the Baylor College of Medicine, Human Genome Sequencing Center (Houston, TX) and SeqWright, Inc. (Houston, TX). Feedback from personnel involved in these beta tests will be incorporated into the next phase of technology development.

描述(申请人提供)：VisiGen BioTechnologies，Inc.正在开发一种测序系统，在该系统中，聚合酶和核苷酸一起作为DNA碱基身份的直接分子传感器。更具体地说，该技术检测修饰的核苷酸和荧光修饰的聚合酶之间的相互作用。当核苷酸被掺入不断增长的DNA聚合物中时，能量从聚合酶中被激发的供体荧光团转移到核苷酸中的受体荧光团，刺激直接实时检测的碱基特异性掺入信号的发射。包括单分子检测、荧光分子化学、计算生物化学和生物分子工程在内的尖端技术结合在一起，创造了这个新的测序系统。DNA样本将在大规模并行阵列中进行处理，这将使大型基因组能够在不到一天的时间内测序，成本约为1000美元，并允许足够的过采样来产生冗余数据，从而将错误降至最低。目前正在开发该系统，以识别病原体(或其变体)，并进行全面的基因组分析。 证明了VisiGen技术的原理：使用一个固定的、供体标记的聚合酶和两个不同的受体标记的dNTP，在单分子水平上实时检测到了序列信息。鉴于我们联邦资助的努力取得了成功，进一步开发VisiGen系统以实时对单DNA分子阵列进行测序显然是必要的。拟议研究的具体目标是改进测序化学，并设计、建造和测试下一代单分子DNA测序仪。这台名为Alpha-1的仪器将被放置在VisiGen上，并加速技术开发。该仪器将用于贝勒医学院、人类基因组测序中心(德克萨斯州休斯顿)和SeqWright，Inc.(德克萨斯州休斯顿)的研究人员进行贝塔测试。参与这些Beta测试的人员的反馈将被纳入下一阶段的技术开发。