The anticipated demand for DNA-based genetic information, in both clinical and basic research laboratories, is virtually unlimited. The biochemical manipulations for DNA analysis are well characterized but have not been assembled into a simple automated system. This proposal will develop a novel, fully integrated technology for DNA processing and analysis. All the steps for extracting genotype information by the polymerase chain reaction (PCR) will occur within a single self-contained system. Both the biochemical and electronic components of the system will be fabricated on a silicon wafer substrate, yielding a micromechanical integrated DNA analysis technology or MIDAT. An enormous range of mechanical and electrical devices can be constructed on silicon using advanced micromechanical fabrication techniques. Since the characteristics of silicon are well understood, designs for components will be generated, tested, and replicated using computer-aided design (CAD) programs. Silicon fabrication of the MIDAT technology will allow for rapid design modification and miniaturization. The low marginal cost of mass-produced silicon devices makes the system disposable, thereby reducing the risk of sample cross-contamination and handling error. By using a reduced quantity of reaction components and a single-use liquid handling system, microscale genotyping will result in significantly lowered sample processing costs and increased sample throughput. Following design and testing of the basic MIDAT components, single silicon wafers will be constructed containing multiple (10 to 1000) parallel processing units. Each unit will PCR amplify a DNA sample and extract computer- readable genotype data. This proposal has five specific goals: l) Demonstration of a liquid handling system using micromachined channels in silicon. Process steps will include the movement, mixing, and distribution of nanoliter drops in silicon. 2) Demonstration of micro-scale genotyping biochemistry. Standard molecular biology DNA reactions, including PCR, will be performed on a nanoliter scale. The reactions will occur entirely within a silicon wafer format. 3) Demonstration of micro-scale DNA size separation and detection. DNA samples will be size-fractionated on an electrophoresis system in silicon. DNA products will be visualized in the system by fluorescent photodetection or radioactive detection. 4) Integration of micromachined components on a single substrate. Liquid handling, electrophoresis, and electronic detector components will be coupled in an integrated silicon- based format. 5) High-throughput DNA genotype analysis. An integrated PCR processing design will be arrayed as parallel units on a single silicon wafer. The number of parallel PCR processing units per wafer will be maximized, and on-chip circuitry developed for process control. A large number of simultaneous PCR genotypings (up to 1000 per wafer) will be performed to test the system and identify improvements in the technology.
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