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）：具有高精度和速度和低成本的人类基因组对人类基因组进行测序的能力对于新兴医学的新兴领域至关重要。为了应对这一需求，我们的研究团队开发了一种新型的在固体表面上划分的DNA测序方法，该方法已被认为是一种成功的解密DNA序列的新范式。在此赠款应用中，我们将使用分子工程方法将成功的SBS策略提升到一个新的水平，通过使用荧光可逆终结器将其调整以进行单分子测序。模板DNA分子将在玻璃表面附着在玻璃表面上，该玻璃表面通过钉蛋白酶的共价附着的条件在玻璃表面上附着，在这种情况下，多达10亿个明显分离的单分子附着在载玻片上，并通过存在可裂解的荧光部分的位置注册。然后，将使用具有适当可裂解的荧光团的可逆封闭核苷酸进行SBS。我们还制定了一种步行策略，该策略允许多次重复使用模板以增加SBS读取长度。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，以在每个SBS的每个周期中大规模检测单分子。最后，我们设计了许多DNA库的构造方法，它们避免了放大，并且与单分子SBS方法兼容的配对末端测序策略。这将使我们能够使用正在进行的生物医学研究合作中的实际基因组DNA，cDNA和其他模板测试该系统。在单分子分辨率下将十亿个DNA模板固定在芯片上，即使30至50个碱基读数也将在单个芯片上覆盖整个人类基因组。公共卫生相关性：实现对个性化医学的需求鼓励了能够以低成本准确性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，我们将通过合成和顺序步行方法成功测序的概念与使用单分子的能力相结合。后者避免了测序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，最耗时的部分之一，并且可能导致DNA序列中的不良偏见。在单分子分辨率下固定在芯片上的十亿个DNA分子，即使读取30或50个碱基的读取长度也将提供能够在单个测序芯片上以高精度对整个人类基因组进行测序的能力。

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。