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.
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