MICROFLUIDS, MICROREACTIONS, AND MICROSEPARATIONS

DNA 基因分型和测序的分析方法与多步骤处理系统相连，从 DNA 源材料开始提取遗传信息。在传统方法中，处理系统对批量的微升体积反应样品以及匹配的液体处理装置和电泳分析设备进行操作。项目 2 将提供组件级设计并测试我们改进的纳升体积处理组件，用于 DNA 测序和基因分型。为了实现在单个设备上执行复杂的多步骤任务的目标，需要离散滴样品处理和信息控制。新的或改进的制造技术也将根据需要进行审查。该项目中开发的所有组件都保留了最终集成到项目 1 的复杂系统中的目标。项目 2 有三个具体目标。 1) 开发纳升注射和移动系统。我们将构建和测试稳健且灵活的注射和移动系统的组件，该系统可以处理体积范围为 10 nl 至 500 nl 的多种液体样品。我们成功的单滴和二元滴系统的注射和移动系统将被修改并组装成多样品液体处理程序。每个离散的液滴都将被独立处理和操纵。 2）开发多个独立的反应室。我们将构建和测试反应室，以实现反应室组件之间的最佳表面相互作用、温度分布和热串扰。一项重大工作将涉及开发控制软件和可重复的反应条件，以实现高质量 DNA 测序。 3) 展示高分辨率分离。我们将改进现有的凝胶电泳系统（大约10-50 bp分辨率，500 bp长度），以接近常规大规模DNA测序的质量（1 bp分辨率，1000 bp长度）。将强调现有荧光标记和检测方法的优化。额外的工作将检查凝胶电泳基质的修饰和替代处理生物化学的效果。

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.