Miniaturized Integrated DNA Analysis Systems

我们的目标是开发小型化的集成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。这些能力将有助于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。毛细管电泳道和微流控样品制备组件将采用光刻和湿法化学刻蚀技术在玻璃基板上制作。通过在直径为10-100微米的非常小的通道上施加高场，并利用微流控样品加载，将实现高速毛细管凝胶电泳感。通过生产高密度径向毛细管电泳道阵列和使用旋转共聚焦荧光扫描仪，将实现高通量和灵敏度。通过将DNA样品准备、样品传输、样品注入和电泳分析集成到芯片上，将实现低成本和强大的过程。这些长期目标将通过完成以下具体目标来实现：(1)96通道径向测序微板和方法将针对DNA测序进行优化，目标是在&lt；30分钟内实现每条通道500个Phred 20个碱基的分离。(2)研制和测试了可在单个微板上扩增和分析96个基因组DNA样品的PCR-CAE微板。(3)将发展微流控固相方法，在100-300NL反应器中进行芯片上热循环(TC)，产生DNA延伸反应，然后在芯片上进行延伸片段纯化，并加载到微制造CE通道上进行测序。(4)开发和测试PC-CAE微板的芯片设计和热循环平台，该平台可以在一个微板上制备、纯化和分析96个测序样品。(5)开发一种BC-芯片，该芯片可以与GFP等标记一起进入大肠杆菌。在短暂生长以实现筛选后，大肠杆菌细胞库将被引入芯片并通过细胞分选器进行鉴定，并将单个转化细胞送入单细胞聚合酶链式反应反应器。在对插入片段进行PCR扩增后，将使用固相捕获化学方法固定模板并提纯后续延伸产物以进行测序分析。单次运行该芯片分析96个克隆将产生大约IX个覆盖范围。

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.