INTEGRATED DNA GENOTYPING AND SEQUENCING MICROSYSTEMS

集成 DNA 基因分型和测序微系统

• 批准号: 6583200
• 负责人: David T. BURKE
• 金额: $ 16.41万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-10 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6583200
• 关键词: DNA; biomaterial development /preparation; biomedical equipment development; capillary electrophoresis; fluid; gel electrophoresis; genotype; method development; miniature biomedical equipment; nucleic acid quantitation /detection; nucleic acid sequence

Analysis methods for DNA genotyping and sequencing are linked into multiple-step processing system that begin with DNA source material and extract genetic information. In conventional methods, the processing system operates on batches of microliter-volume reaction samples, together with matched-liquid handling devices and electrophoretic analysis equipment. Project 1 will provide a systems-level approach to evaluate the integrated nano-liter volume components and fabrication technologies developed in the Program Project. The success of microfabricated components will be measured by their ability to work as an autonomous multi-step processing system. Project 1 has four Specific Aims: 1) Develop the microfluidics necessary for integrated nanoliter-scale DNA analysis. Microfluidic components will be used to construct an integrated system that can perform and monitor multiple parallel reactions. An interface with microliter-volume source samples will also be addressed. 2) Demonstrate sequencing and genotyping reactions in an integrated system. Microfluidic devices will be optimized for use with Sanger sequencing or genomic amplification reactions and denaturing gel electrophoresis. The system-level device will perform and monitor polymerase reactions, post reaction treatment, and electrophoretic band migration, with minimal operator intervention. 3) Demonstrate high-quality genomic DNA sequencing in an integrated systems. The resolution of the integrated nanoliter-volume system will be improved to match the level of existing microliter-scale integrated nanoliter-volume system will be improved to match the level of existing microliter-scale technologies. This will include improved fluorescence detectors (with Project 3) and higher resolution gels (with Project 2). Successful completion of this aim will result in a device that can obtain 500-1000 bp of sequence from a sample of template DNA. 4) Demonstrate complex sequencing strategies in an integrated device. Complex sequencing strategies that obtain maximal information from a single DNA sample, such as bi-directional sequencing or simultaneous restriction digestion, will be added to the integrated sequencing device. Issues of quality control, accuracy, and data reproducibility will also be examined.

用于DNA基因分型和测序的分析方法被连接到多步骤处理系统中，该多步骤处理系统开始于DNA源材料并提取遗传信息。在常规方法中，处理系统与匹配的液体处理装置和电泳分析设备一起对微升体积的反应样品的批次进行操作。项目1将提供一个系统级的方法来评估集成的纳升体积组件和制造技术开发的计划项目。微加工组件的成功将通过其作为自主多步处理系统的工作能力来衡量。项目1有四个具体目标：1）开发集成纳升规模DNA分析所需的微流体。微流控组件将用于构建一个集成系统，可以执行和监测多个并行反应。与微升体积源样品的接口也将得到解决。2)在集成系统中演示测序和基因分型反应。微流体装置将被优化以用于桑格测序或基因组扩增反应和变性凝胶电泳。系统级器械将执行并监测聚合酶反应、反应后处理和电泳条带迁移，操作员干预最少。3)在集成系统中展示高质量的基因组DNA测序。集成纳升体积系统的分辨率将被改进以匹配现有微升规模集成纳升体积系统的水平。这将包括改进的荧光检测器（项目3）和更高分辨率的凝胶（项目2）。成功完成这一目标将导致可以从模板DNA样品获得500-1000 bp序列的装置。4)在集成设备中演示复杂的测序策略。从单个DNA样品获得最大信息的复杂测序策略，例如双向测序或同时限制性消化，将被添加到集成测序设备中。质量控制，准确性和数据再现性的问题也将被检查。