MINIATURIZED INTEGRATED DNA ANALYSIS SYSTEMS

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The goal of this project is to develop Miniaturized Integrated DNA Analysis Systems (MIDAS) on silica substrates or chips that will provide higher- speed, higher throughput DNA sequencing an mapping capabilities at reduced cost. The 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. Fluid access is provided by holes micromachine in the glass substrates, and electrical contact will be provided by vacuum deposited metal films. High speed separations will be achieved by applying high- fields to very small 10 X 50-mum cross section channels in the chips 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 devices, sample injection, and electrophoretic analysis onto the chips. These long- term goals will be achieved by completing the following specific aims: (1) Miniaturized capillary electrophoresis arrays will be photolithographically fabricated on glass substrates. Their design, fabrication and operation will be optimized for performing DNA sequencing and fragment sizing separations using conventionally prepared samples. Further evaluation of these MIDAS-CAE chips will be performed through STR allele analyses and DNA sequencing studies of phycobiliprotein lyase genes. (2) We will develop software for high speed sequence data collection, data reduction, base calling and trace editing that is tayored to the unique capabilities of MIDAS chips. (3) A submicroliter PCR reactor will be fabricated on silica chips and the parameters for amplifying DNA in sub-muL volumes will be determined. The small size of this device should permit significant reduction in the thermal cycling times. Microfluidic methods will be developed to transport and inject amplified DNA samples on CE channels. (4) Once these MIDAS-PCR chips are developed, they will be used to perform automated STR analyses and transposon mapping experiments. (5) In second- generation experiments, components for synthesizing DNA extension fragments from ss-DNA templates will be integrated onto these chips and microfluidic devices and methods will be developed for injecting these fragments into channels for sequencing. Microfluidic methods will also be developed for performing thermal cycling to produce DNA extension reactions from smaller amounts of DNA template followed by on-chip injection and separation. (6) Initial testing of these MIDAS-sequencing and MIDAS-thermal cycling chips will be performed through DNA sequencing of phycobiliprotein lyase genes. (7) Once the fabrication and operation of these MIDAS chips are optimized in the above pilot studies, we will further test and develop them by performing transposon-based DNA mapping and sequencing in collaboration with the LBL Human Genome Center.