MINIATURIZED INTEGRATED DNA ANALYSIS SYSTEMS

该项目的目标是开发小型化集成 在微孔板或芯片上的DNA分析系统， 更高速、更高通量DNA测序和片段化 以更低的成本实现尺寸调整能力。 毛细管电泳 微流体特征将在玻璃基板上制造， 使用光刻和化学蚀刻以及热 结合以在玻璃夹层结构内制造通道。 高速分离将通过施加高场来实现 到非常小的20 × 60 μ m横截面通道 微孔板和利用短的分离距离。 高 通过生产高密度的 独立分析系统。 低成本将通过 使用较低体积的试剂， 样品制备，分析阶段之间的样品运输， 样品注入和电泳分析到芯片上。 这些长期目标将通过完成 具体目的如下：（1）毛细管阵列电泳 能够分析96个DNA测序样品的微孔板将 在玻璃衬底上光刻制造。 的 优化设计、制造、操作和检测 使用常规（芯片外）制备的DNA测序样品。 (2)一系列高速四色共焦芯片扫描仪 将被设计，建造和优化，以检测96个或更多 芯片上的分离通道，采样率至少为10 Hz 每个通道的所有四种颜色。(3)A小于或等于穆尔 热反应堆将在硅芯片上制造， 小体积快速PCR扩增DNA的方法将是 开发 微流体方法将被开发用于运输 并有效地将扩增的DNA样品注射到单个CE上， 渠道(4)用于PCR的芯片设计和热循环仪平台- 然后将开发和测试CAE微孔板， 在单个微孔板上扩增并分析96个样品。(5)一旦 可以使用单独的集成热反应器， 微流控和固相方法将被开发用于 进行芯片上热循环（TC）以产生DNA延伸， 从少量的DNA模板反应，然后在芯片上 分析. (6)用于TC的芯片设计和热循环平台 然后将对CAE微孔板进行优化和测试， 在单个微孔板上制备并分析96个测序样品。

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.