Single-Molecule DNA Sequencing Using Charge-Switch dNTPs

使用电荷开关 dNTP 进行单分子 DNA 测序

• 批准号: 6708517
• 负责人: JOHN G. K. WILLIAMS
• 金额: $ 85万
• 依托单位: LI-COR BIOSCIENCES, INC.
• 依托单位国家: 美国
• 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
• 来源: https://reporter.nih.gov/project-details/6708517
• 关键词: DNA directed DNA polymerase; deoxyribonucleoside triphosphate; high throughput technology; nanotechnology; nucleic acid sequence; pyrimidine nucleotides; technology /technique development

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.

产品说明：（由申请人提供）该计划项目与LI-COR于1998年开始的一项努力有关，该努力旨在开发一种用于对具有非常长读段的单个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。 一个成功的系统将是革命性的速度，读取长度，成本和最小化的实验室基础设施。 整个基因组将从单个基因组DNA样品测序而无需克隆或扩增，并且长读段不仅能够进行从头基因组测序，而且自动提供单倍型信息。 我们的目标是每秒500个原始碱基调用的每仪器吞吐量，具有低错误率（每10，000个成品碱基1个错误，5倍覆盖率）。 试剂和流动池的制造成本最初的目标是每成品（5x）底座约0.001美分，有可能奠定技术框架，使未来能够显著降低额外的成本。 并行仪器和图像分析的发展是独立于本计划项目资助。 该计划有三个目标： 1)设计、制造和评估多通道流动池，这些流动池能够实现DNA模板的珠对接;试剂的流体控制（包括聚合酶和修饰的核苷酸底物）;以及从完整的γ-磷酸盐标记的核苷酸中释放的标记的焦磷酸盐的电荷切换分配。 2)设计、合成和评估四种修饰的核苷酸类型（A、C、G、T），其中这种修饰涉及将具有适于单分子检测的荧光物理学的荧光染料经由各种连接臂构型连接到核苷酸的γ-磷酸以及将电荷部分（例如+2电荷）连接到核苷酸碱基。 3)制备、表达、纯化和筛选突变体聚合酶文库以进化适合于掺入电荷转换核苷酸底物的聚合酶，其核苷酸掺入速率和保真度适合于与多通道流动池一起满足通量目标。