Modular Microfluidic System for Automated DNA Sequencing

描述（由申请人提供）： 我们正在开发一种新型微流体系统，该系统由模块化单元组成，这些单元由PMMA和PC等聚合物制成，使用金属母版对微型零件进行热压纹。每个单元具有离散功能，并使用毛细管互连。最终形式的集成系统将执行以下功能：输入DNA的PCR扩增、PCR产物的纯化、使用染料终止剂化学的循环测序、使用固相可逆固定化（SPRI）的测序梯的纯化和DNA产物的电泳分选。所有这些过程步骤可以在大约30分钟内完成（16通道）。电泳芯片的荧光读数将使用近红外激光扫描显微镜完成。该显微镜不仅收集稳态发射，而且还收集时间分辨数据。使用双色、四寿命方法，荧光检测器将具有处理8个光谱学上独特的荧光探针的能力，这将允许在同一凝胶道中同时处理两个不同的模板。热循环仪将扩增DNA并以16秒/循环的速度进行循环测序，消耗约150 nL试剂，通过减少试剂消耗来最大限度地降低测序成本。纯化芯片将进行毛细管电色谱，用于PCR产物分离，并在PC表面上进行SPRI，PC表面通过UV光活化以产生表面羧酸酯基团。电泳芯片将包含阴离子介孔膜，以允许测序梯的样品预浓缩，从而有助于检测。标记染料将由适当制备的酞菁（Pc）组成，酞菁（Pc）含有水溶性基团，也可以改变基础Pc的吸收特性，以匹配双色显微镜的激光波长（680和780 nm）。为了改变荧光寿命，将不同的金属掺入大环的核心。为了评估用于DNA测序的集成系统的性能，我们将研究填充的Alu染色体位置之间的基因转换/替换事件以及从基因组中去除这些元件。将对来自地理上不同的人类基因组样本的每个基因座的2,000个等位基因进行PCR，并使用模块化微流体系统直接测序。

DESCRIPTION (provided by applicant): We are developing a novel microfluidic system that is composed of modular units that are fabricated from polymers, such as PMMA and PC, using a metal master to hot emboss microparts. Each unit possesses a discrete function and are interconnected using capillaries. The integrated system, in its final form, will perform the following functions, PCR amplification of the input DNA, purification of the PCR products, cycle sequencing using dye-terminator chemistry, purification of sequencing ladders using solid-phase reversible immobilization (SPRI) and electrophoresis sorting of the DNA products. All of these process steps can be completed in about 30 mins (16-channels). Fluorescence readout of the electrophoresis chip will be accomplished using a near-IR laser scanning microscope. This microscope not only collects steady-state emission, but also time resolved data. Using a two-color, four-lifetime approach, the fluorescence detector will possess the ability to process 8-spectroscopically unique fluorescent probes, which will permit processing two different templates simultaneously in the same gel tract. The thermal cycler will amplify DNA and perform cycle sequencing at a speed of 16 s/cycle and consume approximately 150 nL of reagents, minimizing sequencing cost by reducing reagent consumption. The purification chips will perform capillary electrochromatography for PCR product isolation and SPRI on PC surfaces activated by UV light to create surface carboxylate groups. The electrophoresis chip will contain an anionic mesoporous membrane to allow sample pre-concentration of the sequencing ladders to aid in detection. Labeling dyes will consist of appropriately prepared phthalocyanines (Pc) that contain water solubilizing groups that also alter the absorption properties of the base Pc to match the lasing wavelengths of the two-color microscope (680 and 780 nms). To change the fluorescence lifetimes, different metals will be incorporated into the core of the macrocycle. To evaluate the performance of the integrated system for DNA sequencing, we will look at gene conversion/replacement events between filled Alu chromosomal locations as well as the removal of these elements from the genome. PCR of 2,000 alleles for each locus from geographically diverse human genome samples will be undertaken and sequenced directly using the modular microfluidic system.