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Single-Molecule DNA Sequencing Using Charge-Switch dNTPs

使用电荷开关 dNTP 进行单分子 DNA 测序

基本信息

批准号：
6904636
负责人：
JOHN G. K. WILLIAMS
金额：
$ 107.7万
依托单位：
LI-COR BIOSCIENCES, INC.
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-06-18 至 2007-04-30
项目状态：
已结题

项目摘要

DESCRIPTION: (provided by applicant) This Program Project is related to an effort at LI-COR begun in 1998 to develop a system for de novo sequencing of single DNA molecules with very long reads. In particular, the Program Project will further the development of reagents and microfluidics flowcells for the system. A successful system would be revolutionary with respect to speed, read length, cost and minimized laboratory infrastructure. An entire genome would be sequenced from a single genomic DNA sample without cloning or amplification, and the long reads not only enable de novo genome sequencing, but automatically provide haplotype information. We are targeting a per-instrument throughput of 500 raw base calls per second with low error rates (1 error per 10,000 finished bases with 5x coverage). Manufacturing cost for reagents and flowcells is initially targeted to be about 0.001 CENTS per FINISHED (5x) base, with the potential of laying the technological framework to enable future significant additional cost reductions. Concurrent instrumentation and image analysis developments are funded independently of this Program Project. The proposal has three Program Goals: 1) Design, fabricate, and evaluate multichannel flowcells that enable bead-docking of DNA templates; fluidic control of reagents (including polymerases and modified nucleotide substrates); and charge-switched partitioning of released labeled pyrophosphates from intact gamma-phosphate-labeled nucleotides. 2) Design, synthesize, and evaluate four modified nucleotide types (A,C,G,T) whereby such modification involves attaching a fluorescent dye with photophysics suitable for single molecule detection via various linker arm configurations to the gamma-phosphate of the nucleotide as well as the attachment of a charge moiety (e.g. +2 charge) to the nucleotide base. 3) Preparation, expression, purification and screening of mutant polymerase libraries to evolve a polymerase that is suitable for incorporating the charge-switch nucleotide substrates with a nucleotide incorporation rate and fidelity as appropriate for meeting the throughput goals in conjunction with the multichannel flowcells.

描述：(由申请人提供)本计划项目与1998年开始的Li-COR的一项工作有关，该项目旨在开发一种用于对具有很长读数的单DNA分子进行从头测序的系统。特别是，该计划项目将进一步开发用于该系统的试剂和微流体流动池。一个成功的系统将在速度、阅读长度、成本和最大限度地减少实验室基础设施方面具有革命性。整个基因组将从单个基因组DNA样本中测序，而不需要克隆或扩增，而且长的读数不仅能够从头开始基因组测序，而且自动提供单倍型信息。我们的目标是每台仪器的吞吐量为每秒500个原始基本调用，错误率较低(覆盖范围为5倍的每10,000个已完成基本调用中有1个错误)。试剂和流动电池的制造成本最初的目标是每个成品(5倍)基数约0.001美分，有可能奠定技术框架，使未来能够显著地进一步降低成本。同时进行的仪器和图像分析开发的资金独立于本计划项目。该提案有三个计划目标： 1)设计、制造和评估多通道流动池，以实现DNA模板的珠对接；试剂(包括聚合酶和修饰的核苷酸底物)的流体控制；以及从完整的伽马磷酸标记核苷酸中电荷开关分配释放的标记焦磷酸盐。 2)设计、合成和评价四种修饰的核苷酸类型(A、C、G、T)，其中这种修饰包括通过不同的连接臂配置将适合于单分子检测的具有光物理的荧光染料连接到核苷酸的伽马磷酸上，以及将电荷部分(例如+2电荷)连接到核苷酸碱基上。 3)突变聚合酶文库的制备、表达、纯化和筛选，以进化适合于结合电荷开关核苷酸底物的聚合酶，该聚合酶具有适当的核苷酸参入率和保真度，以满足与多通道流动细胞结合的吞吐量目标。