MINIATURIZED INTEGRATED DNA ANALYSIS SYSTEMS

本项目的目标是开发小型化集成DNA分析 系统（MIDAS）的二氧化硅基板或芯片，将提供更高的- 速度更快、通量更高的DNA测序和绘图能力， 成本毛细管电泳和微流控特征将是 使用光刻和化学蚀刻在玻璃衬底上制造 与热粘合一起在玻璃夹层内制造通道 结构.流体通道是由玻璃上的微机械孔提供的 基板和电接触将提供真空沉积 金属薄膜高速分离将通过应用高速- 场到芯片中非常小的10 × 50 μ m横截面通道， 利用短的分离距离。高吞吐量将通过以下方式实现 产生独立分析系统的高密度阵列。低成本 将通过使用较低体积的试剂和 将DNA样品制备、装置之间的样品运输 样品注入和电泳分析到芯片上。这些长- 通过完成以下具体目标来实现本学期的目标：（1） 微型化毛细管电泳阵列将在毛细管电泳仪上进行。 制造在玻璃衬底上。它们的设计、制造和操作 将被优化用于进行DNA测序和片段大小测定 使用常规制备的样品进行分离。进一步评价 这些MIDAS-CAE芯片将通过STR等位基因分析和DNA 藻胆蛋白裂解酶基因的测序研究。(2)我们将开发 用于高速序列数据采集、数据简化、基础 调用和跟踪编辑，这是tayored的独特功能， MIDAS芯片。(3)将在二氧化硅上制作亚微升PCR反应器 用于扩增亚μ L体积的DNA的芯片和参数将是 测定该装置的小尺寸应允许显著的 减少热循环时间。微流体方法将是 开发用于在CE通道上运输和注射扩增的DNA样品。 (4)一旦这些MIDAS-PCR芯片被开发出来，它们将被用来执行 自动STR分析和转座子定位实验。(5)在第二- 生成实验，用于合成DNA延伸片段的组分 将被整合到这些芯片上， 将开发用于将这些碎片注入到 渠道进行排序。微流体方法也将被开发用于 进行热循环，从较小的 量的DNA模板，随后进行芯片上注射和分离。（六） 这些MIDAS测序和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芯片的制造和操作得到优化 在上述试验研究中，我们会进一步测试和发展这些研究， 合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.