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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

基本信息

批准号：
8091384
负责人：
JINGYUE JU
金额：
$ 55.81万
依托单位：
COLUMBIA UNIV NEW YORK MORNINGSIDE
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-01 至 2013-06-30
项目状态：
已结题

项目摘要

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于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固体表面DNA合成测序（SBS）的新方法，该方法被认为是破译DNA序列的成功新范例。在本次拨款申请中，我们将使用分子工程方法，通过使用荧光可逆终止子使其适应单分子测序，将我们成功的 SBS 策略提升到一个新的水平。模板 DNA 分子将附着在通过共价连接 PEG 引物修饰的玻璃表面上，其中多达 10 亿个清晰分离的单分子附着在载玻片上，并通过可裂解荧光部分的存在记录它们的位置。然后使用可逆封闭核苷酸和一组适当的可裂解荧光团进行 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，以创建具有增强型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分子。最后，我们设计了多种避免扩增的DNA文库构建方法以及与单分子SBS方法兼容的双端测序策略。这将使我们能够使用来自正在进行的生物医学研究合作的真实基因组 DNA、cDNA 和其他模板来测试该系统。由于十亿个 DNA 模板以单分子分辨率固定在芯片上，即使是 30 到 50 个碱基读数也能在单个芯片上以良好的覆盖度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、高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，我们将成功的合成测序和序列行走方法的概念与利用单分子的能力结合起来。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 序列中出现不良偏差。由于十亿个 DNA 分子以单分子分辨率固定在芯片上，即使读取长度为 30 或 50 个碱基，也能在单个测序芯片上高精度地对整个人类基因组进行测序。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

PEG-labeled nucleotides and nanopore detection for single molecule DNA sequencing by synthesis.

DOI：
10.1038/srep00684
发表时间：
2012
期刊：
SCIENTIFIC REPORTS
影响因子：
4.6
作者：
Kumar, Shiv;Tao, Chuanjuan;Chien, Minchen;Hellner, Brittney;Balijepalli, Arvind;Robertson, Joseph W. F.;Li, Zengmin;Russo, James J.;Reiner, Joseph E.;Kasianowicz, John J.;Ju, Jingyue
通讯作者：
Ju, Jingyue

7-(3-Nitro-phen-yl)-9,10-dihydro-7H-benzo[h]cyclo-penta-[b]quinolin-8(11H)-one.

7-(3-硝基-苯-基)-9,10-二氢-7H-苯并[h]环五-[b]喹啉-8(11H)-酮。