MINIATURIZED INTEGRATED DNA ANALYSIS SYSTEMS

本项目的目标是开发微型化集成DNA分析 二氧化硅基板或芯片上的系统(MIDAS)将提供更高的- 速度、更高吞吐量的DNA测序和作图能力降低 成本。毛细管电泳法和微流控技术将 利用光刻和化学腐蚀技术在玻璃基板上制作 与热键合一起在玻璃夹层内制造沟槽 结构。液体通道由玻璃上的微孔机械提供 衬底，电接触将通过真空沉积提供 金属薄膜。高速分离将通过应用高速分离器实现 场到芯片中非常小的10 X 50微米横截面通道 利用短间隔距离。高吞吐量将通过以下方式实现 生产高密度的独立分析系统阵列。低成本 将通过使用较少的试剂和通过 将DNA样品的制备、设备之间的样品传输、 样品注入，并在芯片上进行电泳分析。这些长长的- 通过完成以下具体目标来实现任期目标：(1) 微型化的毛细管电泳阵列为光刻技术 在玻璃基板上制造。它们的设计、制造和运营 将针对执行DNA测序和片段大小调整进行优化 使用常规制备的样品进行分离。进一步评估 这些MIDAS-CAE芯片将通过STR等位基因分析和DNA进行 藻胆蛋白裂解酶基因的测序研究。(二)发展 用于高速序列数据采集、数据还原、数据库软件 调用和跟踪编辑，这是专为 迈达斯薯片。(3)在二氧化硅上制作亚微升聚合酶链式反应反应器 芯片和用于扩增亚单位体积DNA的参数将是 下定决心。该设备的小尺寸应该允许显著 减少热循环时间。微流控方法将是 开发用于在CE通道上传输和注入扩增的DNA样本。 (4)一旦这些MIDAS-PCR芯片被开发出来，它们将被用于 自动STR分析和转座子作图实验。(5)每秒- 世代实验，合成DNA延伸片段的成分 来自ss-DNA模板将被集成到这些芯片和微流控芯片上 将开发用于将这些碎片注入到 用于排序的通道。微流控方法也将被开发用于 执行热循环以产生较小的DNA延伸反应 DNA模板量，然后芯片上进样和分离。(6) 这些MIDAS排序和MIDAS热循环芯片的初步测试 将通过藻胆蛋白裂解酶基因的DNA测序进行。 (7)一旦这些MIDAS芯片的制造和运行得到优化 在上述先导研究中，我们将通过以下方式进一步测试和开发它们 协作进行基于转座子的DNA作图和测序 LBL人类基因组中心。

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.