Microbead INtegrated DNA Sequencer (MINDS) System

微珠集成 DNA 测序仪 (MINDS) 系统

• 批准号: 6887951
• 负责人: STEVAN B JOVANOVICH
• 金额: $ 147.6万
• 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/6887951
• 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(申请人提供)：这个合作项目旨在开发一种“微珠集成DNA测序仪”(Minds)，它有效地集成了DNA测序中的所有主要步骤，从文库构建到使用低成本微流控设备的最终序列输出。自动化的MENTS系统将结合三个基本步骤：1)使用微珠菌落技术的文库构建、扩增和选择；2)纳升循环测序样品的准备和纯化；3)基于微制造毛细管阵列电泳(UCAE)的DNA测序片段的分离。文库构建和扩增过程将输入剪切后的大小DNA片段，并构建微珠模板的乳胶聚合酶链式反应扩增文库，每个微珠代表一个DNA片段。然后，单个珠子将在25NL循环测序反应器中进行处理，以产生荧光标记的测序片段，这些片段可以使用芯片上的亲和捕获有效地捕获、浓缩和纯化。然后，这些片段在经过验证的微型制造uCAE测序仪上进行分离和大小调整。 这项提案将把微芯片生物技术公司(MBI)的努力与三个合作学术机构的分包合同结合起来。MBI将开发一个基于微芯片的DNA样品制备纳米级热循环模块的原型，以及一个使用传统外部化学的uCAE测序系统原型的先进旋转扫描仪。然后，这些将被集成在一起，以生产具有25 NL循环测序、样品制备、亲和纯化和uCAE测序阵列的Minds微芯片。当这项工作完成后，到30个月时，MBI将进一步整合Mathies实验室正在开发的基于微珠的库技术，以创建400个准备进行Beta测试的通道Mind系统原型。这些发展将建立在学术合作者共同开发的新方法和战略的基础上，特别是uCAE分离系统和基于微流控的“克隆”方法。加州大学伯克利分校Mathies实验室的分包合同将支持克隆扩增和选择的新微技术的开发，并将这些方法和过程与原型微型测序系统相结合。与Mathies合作，西北大学的巴伦实验室将开发和测试新型DNA分离矩阵，这种矩阵可以轻松地加载到芯片微通道中或从芯片微通道中更换，并提供至少700个碱基的快速、高分辨率分离。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的新方法，以及对MBI生产的集成测序系统进行贝塔测试。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的方法，以及芯片上样本的清理。该项目的目标是在哥伦比亚基因组中心放置一个完全集成的原型Sanger测序系统的测试版，并展示其以100倍的成本进行基因组测序和重新测序的能力，通过生产超过1.5千兆碱基的猎枪序列，吞吐量约为700万个碱基/天/机器。 MENTS系统将利用成熟的uCAE设备的能力，通过制备与分析要求更匹配的体积的样品来分析亚纳升体积，从而极大地降低鸟枪式测序和重新测序的成本，将循环测序试剂的消耗减少100倍。文库的构建将以基于珠子的形式自动进行，扩增和选择在单一批量反应中全面进行，再次减少试剂消耗和成本。一种为微芯片设计的新型聚合物分离基质已经显示出良好的性能，随着微流控体积的减少，将使基质成本降至最低。有了这些组合创新，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Sanger荧光DNA测序的终极性能。未来的工作将探索开发适合核心实验室和个别实验室的低通量版本。