The goal of this project is to develop Miniaturized Integrated DNA Analysis Systems on microplates or chips that will provide higher-speed, higher throughput DNA sequencing and fragment sizing capabilities at reduced cost. Capillary electrophoresis and microfluidic features will be fabricated on glass substrates using photolithography and chemical etching together with thermal bonding to fabricate channels within glass sandwich structures. High speed separations will be achieved by applying high-fields to very small 20 x 60 mum cross section channels in the microplates and by utilizing short separation distances. High throughput will be achieved by producing high density arrays of independent analysis systems. Low cost will be achieved by working with lower volumes of reagents and by integrating the DNA sample preparation, sample transport between analysis stages, sample injection, and electrophoretic analysis onto the chips. These long-term goals will be achieved by completing the following specific aims: (1) Capillary array electrophoresis microplates capable of analyzing 96 DNA sequencing samples will be photolithographically fabricated on glass substrates. The design, fabrication, operation and detection will be optimized using conventionally (off-chip) prepared DNA sequencing samples. (2) A series of high-speed, four-color confocal chip scanners will be designed, built and optimized for detecting 96 or more separation channels on a chip with at least 10 Hz sampling rates per channel for all four colors. (3) A less than or equal to muL thermal reactor will be fabricated on silica chips and the methods for rapidly PCR amplifying DNA in small volumes will be developed. Microfluidic methods will be developed to transport and efficiently inject the amplified DNA samples on individual CE channels. (4) Chip designs and a thermal cycler platform for PCR- CAE microplates will then be developed and tested that can amplify and analyze 96 samples on a single microplate. (5) Once individual integrated thermal reactors are available, microfluidic and solid-phase methods will be developed for performing on-chip thermal cycling (TC) to produce DNA extension reactions from small amounts of DNA template followed by on-chip analysis. (6) Chip designs and a thermal cycling platform for TC- CAE microplates will then be optimized and tested that can prepare and analyze 96 sequencing samples on a single microplate.
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