A Turn Key Approach to High Throughput DNA Sequencing

高通量 DNA 测序的交钥匙方法

• 批准号: 6789203
• 负责人: Gregory John Kellogg
• 金额: $ 10万
• 依托单位: NETWORK BIOSYSTEMS, INC.
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-24 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6789203
• 关键词: DNA; biomedical automation; biomedical equipment development; biotechnology; capillary electrophoresis; fluid flow; high throughput technology; nucleic acid sequence; physical separation; plastics

DESCRIPTION (provided by applicant): The specific aim of this Phase I proposal is to establish proof of concept for development of an integrated microfluidic DNA sample prep bioMEMS device in order to greatly streamline DNA sequencing time and cost. The objectives to be addressed include: 1. Fabricating a single-use plastic microfluidic bioMEMS device (bioMEMS Card A) that removes salts and unincorporated nucleotides from a cycle sequencing reaction 2. Demonstrating that bioMEMS Card A efficiently removes these contaminants 3. Demonstrating that bioMEMS Card A can deliver a nanoliter sample volume to a glass microfluidic bioMEMS device (bioMEMS plate B) that is part of a commercial DNA sequencer 4. Comparing the sequencing results from (a) - (c) with a comparable sample prepared by ethanol precipitation and analyzed by capillary electrophoresis. This Phase I proposal is intended as part of the larger commercial effort to develop an integrated bioMEMS-based DNA sequencing system based on conventional sequencing methodologies that reduces overall reagent use by at least 100-fold. Through the microscale integration of the component steps, the system would also offer higher throughput and reduced run-to-run variability. The end result will be a commercial system that seamlessly integrates DNA sequencing from sample prep to detection on a microfluidic scale. Several groups have demonstrated microfluidic implementations of the individual steps in the overall DNA sequencing process, including amplification, filtration, and separation. To date, however, the inability to micro-integrate the various component pieces together have prevented these advances from significantly reducing the overall cost/performance of sequencing. The initial challenge is to demonstrate that multiple microfluidic-based sub-systems (designed for different chemistries, operating environments, cost points; and fabricated with different substrate materials) can be successfully integrated. For Phase I, a single use plastic bioMEMS device that replaces the purification step after the cycle sequencing reaction will be developed and demonstrated to interface with existing, multi-use glass-based microfluidic electrophoresis. The benefits of developing an automated, integrated microfluidic DNA sequencing platform include reduced reagent consumption, increased throughput, increased accuracy and faster results. Commercial applications of the product include high throughput DNA sequencing and re-sequencing for comparative and functional genomic studies as well as analysis of human genome sequence variation and diagnostics.

描述（由申请人提供）：该阶段I提案的具体目的是建立用于开发集成微流体DNA样品制备bioMEMS设备的概念验证，以便大大简化DNA测序时间和成本。要解决的目标包括：1。制造一次性塑料微流体bioMEMS装置（bioMEMS卡A），其从循环测序反应中去除盐和未掺入的核苷酸2。证明bioMEMS卡A有效地去除了这些污染物3.证明bioMEMS卡A可以将纳升样品体积递送到作为商业DNA测序仪4的一部分的玻璃微流体bioMEMS装置（bioMEMS板B）。将（a）-（c）的测序结果与通过乙醇沉淀制备并通过毛细管电泳分析的可比较样品进行比较。该I期提案旨在作为更大商业努力的一部分，以开发基于常规测序方法的集成bioMEMS DNA测序系统，该系统将总体试剂使用减少至少100倍。通过组分步骤的微尺度集成，该系统还将提供更高的通量和降低的运行间可变性。最终的结果将是一个商业系统，它将DNA测序从样品制备到微流体规模的检测无缝集成。几个小组已经展示了整个DNA测序过程中各个步骤的微流体实现，包括扩增，过滤和分离。然而，迄今为止，无法将各种组件微集成在一起已经阻止了这些进步显著降低测序的总体成本/性能。最初的挑战是证明多个基于微流体的子系统（设计用于不同的化学，操作环境，成本点;并使用不同的衬底材料制造）可以成功集成。对于第一阶段，将开发一种一次性塑料bioMEMS装置，用于取代循环测序反应后的纯化步骤，并证明其与现有的多用途玻璃基微流体电泳相结合。开发自动化、集成的微流体DNA测序平台的好处包括减少试剂消耗、增加通量、提高准确性和更快的结果。该产品的商业应用包括用于比较和功能基因组研究的高通量DNA测序和重测序，以及人类基因组序列变异分析和诊断。