MICROFLUIDS, MICROREACTIONS, AND MICROSEPARATIONS

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，所述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批次进行操作。项目2将为DNA测序和基因分型提供组件级设计和测试我们改进的纳升体积处理组件。为了实现在单个设备上执行复杂的多步骤任务的目标，将需要离散的液滴样品处理和信息控制。还将根据需要审查新的或改进的制造技术。项目中开发的所有组件都保留了最终纳入项目1复杂系统的目标。项目2有三个具体目标。1)开发一个纳升注射和移动系统。我们将构建和测试一个强大而灵活的注射和移动系统的组件，该系统可以处理10 nl至500 nl体积范围内的多种液体样品。我们成功的单液滴和双液滴系统的进样和移动系统将被修改和组装成多样品液体处理程序。每个离散液滴将被独立处理和操作。2)开发多个独立的反应室。我们将构建和测试反应室，以获得最佳的表面相互作用，温度分布和反应室组件之间的热串扰。一个重要的努力将涉及开发控制软件和可重复的反应条件，高质量的DNA测序。3)演示高分辨率分离。我们将改进现有的凝胶电泳系统（约10-50 bp分辨率，500 bp长度），以接近常规大规模DNA测序（1 bp分辨率，1000 bp长度）的质量。优化现有的荧光标记和检测方法将被强调。另外的努力将检查凝胶电泳基质的修改和替代处理生化的影响。

Analysis methods for DNA genotyping and sequencing are linked into multiple-step processing systems 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 2 will provide component-level design and testing our improved nanoliter-volume processing components for DNA sequencing and genotyping. To fulfill the goal of performing complex multi-step tasks on a single device, discrete drop sample handling and informational control will be required. New or improved fabrication technologies will also be examined as needed. All of the components developed in the Project retain the target of eventual integration in the complex systems of Project 1. Project 2 has three Specific Aims. 1) Develop a nanoliter injection and movement system. We will construct and test components for a robust and flexible injection and movement system that can handle multiple liquid samples in the volume range of 10 nl to 500 nl. Our successful injection and movement systems for single and binary drop systems will be modified and assembled into multiple- sample liquid handling procedures. Each discrete drop will be addressed and manipulated independently. 2) Develop multiple independent reaction chambers. We will construct and test reaction chambers for optimal surface interactions, temperature profiles, and thermal cross talk between reaction chamber components. A significant effort will involve developing control software and reproducible reaction conditions for high quality DNA sequencing. 3) Demonstrate high-resolution separations. We will improve the existing gel electrophoresis system (approximately 10-50 bp resolution, 500 bp length), to approach the quality of conventional large-scale DNA sequencing (1 bp resolution, 1000 bp length). Optimization of the existing fluorescent labeling and detection methods will be emphasized. Additional effort will examine gel electrophoresis matrix modifications and the effect of alternative processing biochemistries.