Microbead INtegrated DNA Sequencer (MINDS) System

微珠集成 DNA 测序仪 (MINDS) 系统

• 批准号: 7111675
• 负责人: STEVAN B JOVANOVICH
• 金额: $ 204.53万
• 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/7111675
• 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）：此协作项目旨在开发“微片集成的DNA Sequencer”（心理），该项目有效地整合了DNA测序中的所有主要步骤，从图书馆构造到最终序列输出利用低成本的微富集式序列输出设备。自动化的思维系统将结合三个基本步骤：1）使用Microbead clony Technologies 2）纳米级循环测序样品制备和纯化，以及3）基于Microbabricated毛细管阵列电泳（UCAE）基于DNA测序片段分离的DNA测序片段分离。库的构建和放大过程将输入剪切的，大小的DNA片段，并在珠子上构建乳液PCR放大模板的放大库，每个珠子代表一个单个DNA片段。然后，将在25 NL循环测序反应器中处理单个珠子，以产生荧光标记的测序片段，这些片段被有效地捕获并使用芯片上亲和捕获进行纯化。然后将片段分离并在经过验证的微型UCAE测序仪上大小。 该建议将结合微芯片生物技术公司（MBI）的努力与三家合作学术机构的分包合同。 MBI将使用常规的外部化学分子开发一种原型的基于微芯片的DNA样品制备纳米级热循环模块，并具有带有原型UCAE测序系统的晚期旋转扫描仪。然后将它们集成以产生带有25 NL循环测序样品制备，亲和力纯化和UCAE测序的阵列的思维微芯片。完成此操作后，到30个月后，MBI将进一步整合Mathies Laboratory开发的基于Microbead的图书馆技术，以创建400个渠道思维系统系统原型，以供β测试。这些事态发展将基于学术合作者，特别是UCAE分离系统和基于珠的微流体“克隆”方法而制定的新方法和策略。 U.C. Mathies Lab的分包合同伯克利将支持开发新的微技术来扩增和选择克隆，并将这些方法和过程与原型的微型测序系统的整合。与Mathies合作，Northwestern的Barron Lab将开发和测试新颖的DNA分离矩阵，这些矩阵很容易加载到芯片微通道中，并提供至少700个基本读数的快速，高分辨率分离。哥伦比亚基因组中心JU LAB的分包合同将支持开发基因组克隆生产的新方法，以及对MBI产生的β-测试β测试的集成测序系统。哥伦比亚集团还将与伯克利集团合作，以改善克隆生产和选择的方法，并进行片上样品清理。项目目标是在哥伦比亚基因组中心放置完全集成的原型Sanger测序系统的Beta版本，并证明其以100倍较低的成本进行基因组测序和重新取代的能力，大约700万个基地/天/通过生产超过1.5千兆子枪序列的机器。 思维系统将通过利用良好建立的UCAE设备的能力来大大降低shot弹枪测序和重新取证的成本-折叠。图书馆的构造将以基于珠子的格式自动化，并以单个大量反应进行扩增和选择，再次降低了试剂的消耗和成本。一个专为微芯片设计的新型聚合分离矩阵已经显示出良好的性能，并且，以及微流体体积减少，可以最大程度地减少矩阵费用。通过这些组合创新，思维系统将使CAE仪器在基因组中心的超高通知实施中的四色Sanger荧光DNA测序接近最终性能。未来的工作将探索适合核心和个体实验室的低通量版本的开发。