MICROFABRICATION OF AN INTEGRATED DNA SEQUENCING SYSTEM

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The anticipated demand for DNA sequence information, in both clinical and basic research laboratories, is virtually unlimited. The biochemical and analytical manipulations for DNA sequencing are well-characterized but have not been assembled into a simple integrated system. Based on established methods, this proposal will develop the sample handling, electrophoresis, detection, and control components for a fully integrated DNA sequencing technology. Complete integration ensures that each sample entering the system will have a unique, dedicated set of equipment, thereby eliminating sample processing bottlenecks. The components will be formed using photolithographic microfabrication techniques on crystalline silicon or glass. Photolithographic fabrication allows components to be designed for compatibility, easy assembly into novel combinations, and inexpensive mass production. Advanced silicon and glass thin-film fabrication methods can make a wide range of mechanical and electronic devices -- ranging from millimeter to submicron in size. Since the characteristics of silicon- based materials are well understood, designs can be rapidly generated, tested, and replicated using computer-aided design software. The proposed work will develop an integrated DNA sequencing system, including sample handling and electrophoretic separation, using only silicon or glass photolithographic techniques. The system (i) will require minimal operator interaction with the sample liquids, (ii) will provide extensive real-time control of biochemical reactions, (iii) will directly generate electronic output data as bands from sequencing electrophoresis gels, and (iv) will be able to incorporate feedback information from samples for use at downstream decision points. The system will operate on nanoliter volumes of liquid samples and include circuitry for control of sample location, temperature, photodetection, and electrophoresis. The project has three specific aims: (1) construction of a miniature high resolution electrophoresis system, (2) fluidic and electronic integration of the sequencing system, and (3) the elimination of sequencing bottlenecks using intelligent systems. The first two aims will demonstrate the basic feasibility of constructing DNA sequencing equipment from solid-state materials. Aim 3 will combine the sequencing-specific components with general liquid sample handling components to produce a fully functional device.