Single Molecule DNA Sequencing by Fluorescent Nucleotide Reversible Terminators

通过荧光核苷酸可逆终止子进行单分子 DNA 测序

• 批准号: 7923389
• 负责人: JINGYUE JU
• 金额: $ 64.63万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7923389
• 关键词: Animal Model; Applications Grants; Bacteria; Biomedical Research; Cells; Chemicals; Chemistry; Cleaved cell; Cloning; Collaborations; Color; Complementary DNA; DNA; DNA Library; DNA Microarray Chip; DNA Sequence; Detection; Development; Devices; Disease; Engineering; Fluorescent Dyes; Genomics; Glass; Goals; Human Genome; Lead; Length; Libraries; Location; Medicine; Methodology; Methods; Microfluidics; Microscope; Molecular; Natural regeneration; Nucleotides; Polymerase; Process; Reaction; Reading; Reagent; Research; Resolution; Slide; Solid; Speed; Surface; System; Testing; Time; Walking; base; cost; design; fluorophore; gene discovery; novel; nucleotide analog; public health relevance; response; single molecule; technology development

DESCRIPTION (provided by applicant): The ability to sequence a human genome with high accuracy and speed, and at low cost, is critical to the emerging field of personalized medicine. In response to this demand, our research team developed the novel method of DNA sequencing-by-synthesis (SBS) on a solid surface, which has been recognized as a successful new paradigm for deciphering DNA sequences. In this grant application, we will use molecular engineering approaches to take our successful SBS strategy to the next level by adapting it for single molecule sequencing using fluorescent reversible terminators. Template DNA molecules will be attached to a glass surface modified by covalent attachment of PEG-primers under conditions where as many as 1 billion clearly separated single molecules are attached to the slide and their location registered by the presence of a cleavable fluorescent moiety. SBS will then be conducted using reversible blocked nucleotides with an appropriate set of cleavable fluorophores. We have also developed a walking strategy that permits re-use of the template multiple times to increase SBS readlength. We will modify a TIRF microscope to create a device with an enhanced microfluidic flow cell platform to permit large-scale detection of single molecules during each cycle of SBS. Finally, we have designed a number of DNA library construction methods that avoid amplification and a paired-end sequencing strategies compatible with the single molecule SBS approach. This will permit us to test the system with real genomic DNA, cDNA and other templates from ongoing biomedical research collaborations. With a billion DNA templates immobilized on a chip at single molecule resolution, even 30 to 50 base reads will cover the entire human genome at good coverage on a single chip. Public Health Relevance: The realization of the need for personalized medicine has encouraged the development of technologies able to sequence the human genome with high accuracy and speed at low cost. To approach this goal, we have combined the concepts of our successful sequencing by synthesis and sequence walking method with the ability to utilize single molecules. The latter avoids the necessity of cloning or otherwise amplifying DNA before sequencing, which is in fact one of the most expensive and time consuming parts of the process, and can lead to undesirable biases in the DNA sequences. With a billion DNA molecules immobilized on a chip at single molecule resolution, even read lengths of 30 or 50 bases will provide the ability to sequence the entire human genome at high accuracy on a single sequencing chip.

描述（由申请人提供）：以高精度、高速度和低成本对人类基因组进行测序的能力对新兴的个性化医疗领域至关重要。针对这一需求，我们的研究团队开发了基于固体表面的DNA合成测序（SBS）新方法，该方法已被认为是破译DNA序列的成功新范例。在这项拨款申请中，我们将使用分子工程方法将我们成功的SBS策略提升到一个新的水平，通过使用荧光可逆终止子将其用于单分子测序。模板DNA分子将附着在通过peg引物共价附着修饰的玻璃表面上，条件是有多达10亿个明确分离的单分子附着在载玻片上，并且它们的位置通过可切割荧光片段的存在来记录。然后，SBS将使用可逆阻断核苷酸与一组适当的可切割荧光团进行。我们还开发了一种遍历策略，允许多次重用模板以增加SBS读长。我们将改进TIRF显微镜，以创建一个具有增强微流控流池平台的设备，以便在SBS的每个周期中大规模检测单个分子。最后，我们设计了一些避免扩增的DNA文库构建方法和与单分子SBS方法兼容的成对端测序策略。这将使我们能够用正在进行的生物医学研究合作中的真实基因组DNA、cDNA和其他模板来测试该系统。以单分子分辨率将十亿DNA模板固定在芯片上，即使30到50个碱基读取也可以在单个芯片上很好地覆盖整个人类基因组。