A Turn Key Approach to High Throughput DNA Sequencing

描述(由申请人提供)：此第一阶段提案的具体目标是为开发集成微流控DNA样品准备生物MEMS设备建立概念证明，以极大地简化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控生物MEMS装置(BioMEMS Card A)，其从循环测序反应中去除盐和未结合的核苷酸2.证明BioMEMS Card A有效地去除这些污染物3.证明BioMEMS Card A可以将纳升样品体积输送到玻璃微流控生物MEMS装置(BioMEMS平板B)，该玻璃微流控生物MEMS装置是商业DNA测序仪的一部分。4.将(A)-(C)的测序结果与乙醇沉淀制备并通过毛细管电泳法分析的可比样品进行比较。这项第一阶段提案旨在作为更大的商业努力的一部分，以开发基于生物MEMS的综合DNA测序系统，该系统基于传统测序方法，将总试剂使用量减少至少100倍。通过对各组成部分步骤的微规模整合，该系统还将提供更高的吞吐量，并减少运行到运行的可变性。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控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控实施，包括扩增、过滤和分离。然而，到目前为止，不能将各种成分片段微整合在一起，阻碍了这些进步显著降低测序的总体成本/性能。最初的挑战是证明可以成功地集成多个基于微流控技术的子系统(为不同的化学成分、运行环境、成本点设计；并使用不同的衬底材料制造)。对于第一阶段，将开发一种一次性使用的塑料生物MEMS设备，以取代循环测序反应后的纯化步骤，并展示与现有的多用途玻璃微流控电泳的接口。开发自动化的集成微流控DNA测序平台的好处包括减少试剂消耗、增加产量、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测序和重新测序，以及人类基因组序列变异和诊断的分析。

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.