Microbead INtegrated DNA Sequencer (MINDS) System

微珠集成 DNA 测序仪 (MINDS) 系统

• 批准号: 6955049
• 负责人: STEVAN B JOVANOVICH
• 金额: $ 175.85万
• 依托单位: MICROCHIP BIOTECHNOLOGIES, INC.
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-30 至 2007-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6955049
• 关键词: capillary electrophoresis; fluid flow; fluorescent dye /probe; genetic library; high throughput technology; microarray technology; nanotechnology; nucleic acid amplification techniques; nucleic acid sequence

DESCRIPTION (provided by applicant): This collaborative project is aimed at the development of a "Microbead INtegrated DNA Sequencer" (MINDS) that efficiently integrates all of the major steps in DNA sequencing, from library construction to final sequence output exploiting low-cost microfluidic devices. The automated MINDS system will combine three fundamental steps: 1) library construction, amplification, and selection using microbead colony technologies 2) nanoliter cycle sequencing sample preparation and purification, and 3) microfabricated capillary array electrophoresis (uCAE)-based separation of DNA sequencing fragments. The library construction and amplification process will input sheared, sized DNA fragments and construct an emulsion PCR amplified library of template on beads, with each bead representing a single DNA fragment. Single beads will then be processed in a 25 nL cycle sequencing reactor to produce fluorescently labeled sequencing fragments that are efficiently captured concentrated and purified using on-chip affinity capture. The fragments are then separated and sized on a proven microfabricated uCAE sequencer. This proposal will combine the efforts of Microchip Biotechnologies Inc. (MBI) with subcontracts to three collaborating academic institutions. MBI will develop a prototype microchip-based DNA sample preparation nanoscale thermal cycling module and an advanced rotary scanner with a prototype uCAE sequencing system using conventional external chemistries. These will then be integrated to produce a MINDS microchip with arrays of 25 nL cycle sequencing sample preparation, affinity purification, and uCAE sequencing. When this has been accomplished, by 30 months, MBI will further integrate the microbead-based library technology being developed by the Mathies laboratory to create 400 channel MINDS System prototypes ready for beta-testing. These developments will build upon novel methods and strategies developed in tandem by the academic collaborators, in particular the uCAE separation system and bead-based microfluidic "cloning" methods. A subcontract to the Mathies lab at U.C. Berkeley will support the development of new microtechnologies for the amplification and selection of clones, and the integration of these methods and processes with prototype microfabricated sequencing systems. In collaboration with Mathies, the Barron lab at Northwestern will develop and test novel DNA separation matrices that are easily loaded into and replaced from chip microchannels, and that provide rapid, high-resolution separations with at least a 700-base read. A subcontract to the Ju lab at the Columbia Genome Center will support the development of new methods for genomic clone production as well as for beta-testing the integrated sequencing systems produced by MBI. The Columbia group will also work with the Berkeley group toward improving methods for clone production and selection, and for on-chip sample clean-up. The project goal is to place a beta version of the fully integrated, prototype Sanger sequencing system at Columbia Genome Center and to demonstrate its capability to perform genomic sequencing and resequencing at 100-fold lower cost and a throughput of about 7 million bases/day/machine by producing over 1.5 gigabase of shotgun sequence. The MINDS system will greatly reduce the cost of shotgun sequencing and resequencing, by exploiting the ability of well established uCAE devices to analyze sub-nanoliter volumes through preparation of samples in volumes more closely matched to the analytical requirements, reducing cycle sequencing reagent consumption by 100-fold. Library construction will be automated in the bead-based format, with amplification and selection performed at full scale in a single bulk reaction, again reducing reagent consumption and cost. A novel polymeric separation matrix designed for microchips already shows good performance and, along with microfluidic volume reductions, will minimize matrix expense. With these combined innovations, the MINDS system will drive CAE instrumentation close to the ultimate performance possible for four-color Sanger fluorescent DNA sequencing in an ultra-high-throughput implementation for genome centers. Future work will explore the development of lower-throughput versions appropriate for core and individual laboratories.

描述（由申请人提供）：该合作项目旨在开发“微珠集成 DNA 测序仪”(MINDS)，它有效地集成了 DNA 测序的所有主要步骤，从文库构建到利用低成本微流体设备的最终序列输出。自动化 MINDS 系统将结合三个基本步骤：1) 使用微珠集落技术进行文库构建、扩增和选择，2) 纳升循环测序样品制备和纯化，以及 3) 基于微型毛细管阵列电泳 (uCAE) 的 DNA 测序片段分离。文库构建和扩增过程将输入剪切后的、调整大小的 DNA 片段，并在珠子上构建乳液 PCR 扩增模板文库，每个珠子代表一个 DNA 片段。然后，单珠将在 25 nL 循环测序反应器中进行处理，以产生荧光标记的测序片段，并使用片上亲和捕获有效捕获、浓缩和纯化这些片段。然后在经过验证的微制造 uCAE 测序仪上分离片段并确定大小。 该提案将把 Microchip Biotechnologies Inc. (MBI) 的努力与三个合作学术机构的分包结合起来。 MBI 将开发基于微芯片的原型 DNA 样品制备纳米级热循环模块和先进的旋转扫描仪，以及使用传统外部化学物质的原型 uCAE 测序系统。然后将它们集成以生产 MINDS 微芯片，其中包含 25 nL 循环测序样品制备、亲和纯化和 uCAE 测序的阵列。当这一目标完成后，到 30 个月，MBI 将进一步集成 Mathies 实验室正在开发的基于微珠的库技术，创建 400 个通道的 MINDS 系统原型，准备进行 Beta 测试。这些发展将建立在学术合作者共同开发的新方法和策略的基础上，特别是 uCAE 分离系统和基于珠的微流体“克隆”方法。与加州大学马蒂斯实验室的分包合同伯克利分校将支持开发用于克隆扩增和选择的新微技术，以及将这些方法和过程与原型微加工测序系统相集成。西北大学的 Barron 实验室将与 Mathies 合作，开发和测试新型 DNA 分离基质，这些基质可以轻松加载到芯片微通道中并从芯片微通道中替换，并提供快速、高分辨率的分离，读取至少 700 个碱基。哥伦比亚基因组中心 Ju 实验室的分包合同将支持开发基因组克隆生产以及对 MBI 生产的集成测序系统进行 beta 测试的新方法。哥伦比亚小组还将与伯克利小组合作，改进克隆生产和选择以及芯片样品清理的方法。该项目的目标是在哥伦比亚基因组中心放置完全集成的桑格测序原型系统的测试版，并展示其以降低 100 倍的成本进行基因组测序和重测序的能力，以及通过生成超过 1.5 GB 的鸟枪序列，实现约 700 万碱基/天/机器的吞吐量。 MINDS 系统将利用成熟的 uCAE 设备分析亚纳升体积的能力，通过制备更接近分析要求的体积样品，从而大大降低鸟枪法测序和重测序的成本，从而将循环测序试剂消耗减少 100 倍。文库构建将以基于珠子的形式实现自动化，并在单个批量反应中全面进行扩增和选择，再次减少试剂消耗和成本。为微芯片设计的新型聚合物分离基质已经表现出良好的性能，并且随着微流体体积的减小，将最大限度地减少基质费用。通过这些综合创新，MINDS 系统将推动 CAE 仪器在基因组中心的超高通量实施中接近四色桑格荧光 DNA 测序的终极性能。未来的工作将探索开发适合核心和个体实验室的低通量版本。