Droplet-Based Digital Microfluidic Genome Sequencing

基于液滴的数字微流控基因组测序

• 批准号: 7140317
• 负责人: RICHARD Barton FAIR
• 金额: $ 24.74万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-01 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7140317
• 关键词: biomedical automation; biotechnology; microfluidics; nucleic acid sequence; technology /technique development

DESCRIPTION (provided by applicant): The overall goal of the R21 section (Year 1) is to demonstrate how existing droplet-based microfluidic electrowetting technology can be modified to perform sequencing by synthesis reaction chemistry based on the introduction of DNA cloned on microbeads into a droplet that is subjected to repeated cycles of nucleotide addition and washing. The aims are: 1) adapt electrowetting technology to demonstrate synthesis reaction chemistry in a bead-based droplet format, including bead washing, bead retention, and transport of enzymatic by-products to remote detection sites; 2) demonstrate a quantitative model for the diffusion reaction equations that describe sequencing on microbeads in droplets and the subsequent amount of light generated; 3) demonstrate through simulation that a single-sided assembly strategy will provide an assembly equivalent to Mouse. The goal of the R33 section is to develop an integrated detection strategy (Years 2-4) for the first genome sequencing demonstration (Years 4-5). The major aims are: 1) adapt electrowetting technology to provide experimental, integrated platforms in Years 2-5 to support research in synthesis reaction chemistry in a bead-based droplet format, including bead washing, retention, on-chip dispensing, and transport of enzymatic by-products to a remote array with an integrated CMOS photosensor array; 2) demonstrate that electrowetting technology can be scaled to a picoliter droplet format, including on-chip dispensing with ¿ 2% volume control, reproducible numbers of beads per dispensed droplet, 100% bead retention during washing operations, controlled merging and splitting of bead droplets and wash droplets, and good reproducibility and control of sequencing-by-synthesis processes (Years 2-5); 3) experimentally determine read length limitations in droplet-based sequencing-by synthesis, and implement software and signal processing strategies and assembly methodologies to improve read lengths and data quality, with a goal to demonstrate 1,000 to 10,000 base pair reads by Year 4; sequence a small genome in Years 4-5.

描述（由申请人提供）：R21部分（第一年）的总体目标是展示如何修改现有的基于液滴的微流体电润湿技术，以通过合成反应化学进行测序，该合成反应化学基于将微珠上克隆的DNA引入到经过重复的核苷酸添加和洗涤循环的液滴中。目标是：1）采用电润湿技术以基于珠子的液滴形式演示合成反应化学，包括珠子清洗、珠子保留以及将酶副产物运输到远程检测站点； 2) 展示扩散反应方程的定量模型，该方程描述了液滴中微珠的测序以及随后产生的光量； 3) 通过模拟证明单面装配策略将提供与鼠标等效的装配。 R33 部分的目标是为首次基因组测序演示（第 4-5 年）开发综合检测策略（第 2-4 年）。主要目标是：1）采用电润湿技术，在第2-5年提供实验性集成平台，以支持基于微珠的液滴格式的合成反应化学研究，包括微珠清洗、保留、芯片上分配以及将酶副产物传输到具有集成CMOS光电传感器阵列的远程阵列； 2) 证明电润湿技术可以扩展到皮升液滴格式，包括具有 2% 体积控制的芯片上分配、每个分配液滴的可重复珠子数量、清洗操作期间 100% 的珠子保留、珠子液滴和清洗液滴的受控合并和分裂，以及合成测序过程的良好再现性和控制（第 2-5 年）； 3) 通过实验确定基于液滴的合成测序中的读长限制，并实施软件和信号处理策略以及组装方法以提高读长和数据质量，目标是到第 4 年展示 1,000 至 10,000 个碱基对读长；在第 4-5 年对一个小基因组进行测序。