Miniaturized Integrated DNA Analysis Systems

我们的目标是开发小型化集成DNA分析系统，以更低的成本提供高速，高通量的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、遗传变异分析以及遗传和传染病诊断。毛细管电泳通道和微流控样品制备组件将使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的感觉将通过施加高场非常小的10-100微米直径的通道，并通过利用微流体样品加载。高通量和灵敏度将通过产生高密度的毛细管电泳通道的径向阵列和通过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、样品注入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针对DNA测序进行优化，目标是在<30分钟的分离中每个泳道>500个Phred 20个碱基。(2)将开发和测试PCR-CAE微孔板，其可以在单个微孔板上PCR扩增和分析96个基因组DNA样品。(3)微流控固相方法将被开发用于执行芯片上的热循环（TC），以产生DNA延伸反应，在100-300 nL的反应器，然后芯片上的延伸片段纯化和加载到微制造的CE通道进行测序。(4)然后将开发和测试用于PC-CAE微孔板的芯片设计和热循环平台，该平台可以在单个微孔板上制备、纯化和分析96个测序样品。(5)最后，将研制出一种能进入E。coli沿着表达GFP等标记。经过短暂的生长，达到选择，E。将大肠杆菌细胞文库引入芯片中，并通过细胞分选仪进行鉴定，将单个转化细胞导入单细胞PCR反应器中。在插入片段的PCR扩增后，固相捕获化学将用于扩增模板并纯化随后的延伸产物用于测序分析。分析96个克隆的该芯片的单次运行将产生约IX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.