Miniaturized Integrated DNA Analysis Systems

我们的目标是开发小型化集成DNA分析系统，以降低成本提供高速，高通量DNA测序和基因分型。这些能力将促进人类和其他基因组的测序、遗传变异的分析以及遗传和传染病的诊断。毛细管电泳通道和微流控样品制备组件将采用光刻和湿化学蚀刻在玻璃基板上制造。高速毛细管凝胶电泳将通过将高场应用于非常小的10-100微米直径的通道和利用微流体样品加载来实现。高通量和灵敏度将通过生产毛细管电泳通道的高密度径向阵列和使用旋转共聚焦荧光扫描仪来实现。通过将DNA样品制备、样品运输、样品注射和电泳分析集成到芯片上，将实现低成本和稳健的过程。这些长期目标将通过完成以下具体目标来实现：(1)96通道径向测序微孔板和方法将优化DNA测序，目标是在<30分钟的分离中每通道获得20个碱基。(2)将开发和测试PCR- cae微孔板，该微孔板可在单个微孔板上扩增和分析96个基因组DNA样本。(3)将开发微流控固相方法，在100-300 nL反应器中进行片上热循环（TC）以产生DNA延伸反应，然后进行片上延伸片段纯化并加载到微制造CE通道上进行测序。(4)然后开发和测试PC-CAE微孔板的芯片设计和热循环平台，该平台可在单个微孔板上制备、纯化和分析96个测序样品。(5)最后，将开发一种BC-chip，该芯片可以与GFP等标记物一起进入大肠杆菌。经过短暂的生长以实现选择，大肠杆菌细胞库将被引入芯片，并通过细胞分选器进行识别，并将单个转化细胞送入单细胞PCR反应器。在插入物的PCR扩增后，固相捕获化学试剂将用于固定模板并纯化随后的延伸产物用于测序分析。单次运行该芯片来分析96个克隆将产生大约IX的覆盖率。

Our goal is to develop Miniaturized Integrated DNA Analysis Systems that provide high-speed, high-throughput DNA sequencing and genotyping at reduced cost. These capabilities will facilitate sequencing of the human and other genomes, the analysis of genetic variation, and genetic and infectious disease diagnosis. Capillary electrophoresis channels and microfluidic sample preparation components will be fabricated on glass substrates using photolithography and wet ch4emical etching. High-speed capillary gel electrophoresis sensations will be achieved by applying high-fields to very small 10-100 mum diameter channels and by exploiting microfluidic sample loading. High- throughput and sensitivity will be achieved by producing high density radial arrays of capillary electrophoresis channels and by using a rotary confocal fluorescence scanner. Low-cost and robust processes will be achieved by integrating the DNA sample preparation, sample transport, sample injection, and electrophoretic analysis onto the chips. These long-term goals will be approached by completing the following specific aims: (1) 96-Channel radial sequencing microplates and methods will be optimized for DNA sequencing with the goal of >500 Phred 20 bases per lane in <30 min separations. (2) PCR-CAE microplates will e developed and tested that can PCR amplify and analyze 96 genomic DNA samples on a single microplate. (3) Microfluidic solid-phase methods will be developed for performing on-chip thermal cycling (TC) to produce DNA extension reactions in 100-300 nL reactors followed by on-chip extension fragment purification and loading onto microfabricated CE channels for sequencing. (4) Chip designs and a thermal cycling platform for PC-CAE microplates will then be developed and tested that can prepare, purify and analyze 96 sequencing samples on a single microplate. (5) Finally, a BC-chip will be developed that can into E. coli along with marker like GFP. After brief growth to achieve selection, the E. coli cell library will be introduced into the chip and run through a cell sorter to identify and route individual transformed cells into single cell PCR reactors. After PCR amplification of the insert, solid phase capture chemistries will be used to immobilize the template and to purify the subsequent extension products for sequencing analysis. A single run of this chip to analyze 96 clones will produce approximately IX coverage.