Single Molecule DNA Sequencing by Fluorescent Nucleotide Reversible Terminators

描述（由申请人提供）：以高准确度和速度以及低成本对人类基因组进行测序的能力对于个性化医疗的新兴领域至关重要。针对这一需求，我们的研究团队开发了固体表面上的DNA合成测序（SBS）新方法，该方法已被公认为破译DNA序列的成功新范例。在这项资助申请中，我们将使用分子工程方法，通过使用荧光可逆终止剂将其用于单分子测序，将我们成功的SBS策略提升到一个新的水平。模板DNA分子将附着到通过PEG引物的共价附着而改性的玻璃表面上，在该条件下，多达10亿个清楚分离的单分子附着到载玻片上，并且它们的位置通过可裂解荧光部分的存在而记录。然后将使用可逆封闭的核苷酸与适当的一组可裂解的荧光团进行SBS。我们还开发了一种步行策略，允许多次重复使用模板以增加SBS读长。我们将修改TIRF显微镜以创建具有增强的微流体流动池平台的设备，以允许在SBS的每个循环期间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方法相容的配对末端测序策略。这将使我们能够测试系统与真实的基因组DNA，cDNA和其他模板正在进行的生物医学研究合作。通过以单分子分辨率将十亿个DNA模板固定在芯片上，即使是30到50个碱基读取也将在单个芯片上以良好的覆盖率覆盖整个人类基因组。 公共卫生相关性：对个性化医疗需求的认识鼓励了能够以低成本高精度和速度对人类基因组进行测序的技术的发展。为了实现这一目标，我们将我们成功的合成测序和序列步移方法的概念与利用单分子的能力相结合。后者避免了在测序之前克隆或以其他方式扩增DNA的必要性，这实际上是该过程中最昂贵和最耗时的部分之一，并且可能导致DNA序列中的不期望的偏差。在以单分子分辨率将十亿个DNA分子固定在芯片上的情况下，即使是30或50个碱基的读取长度也将提供在单个测序芯片上以高准确度对整个人类基因组进行测序的能力。

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.