HIGH THROUGHPUT ELECTROPHORESIS SYSTEM FOR DNA SEQUENCIN

DNA 测序高通量电泳系统

• 批准号: 2209706
• 负责人: JOSEPH BALCH
• 金额: $ 98.66万
• 依托单位: UNIVERSITY OF CALIF-LAWRNC LVRMR NAT LAB
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-30 至 1998-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2209706
• 关键词: charge coupled device camera; computer assisted sequence analysis; electrophoresis; fluorescent dye /probe; lasers; method development; nucleic acid sequence

DESCRIPTION: (Applicant's Abstract) The 2-4 year objective of our DNA sequencing instrumentation program is to assemble a comprehensive facility that can process and analyze at least 3000 clones per 24 hr per sequencing system. In this application we propose to develop one component of this facility--a highly automated electrophoresis system with integrated base calling software, including appropriate real-time analysis tools. When completed, the electrophoresis system will analyze 384 DNA samples simultaneously with a run-to-run cycle time of less than three hours. It will have a peak sequencing rate of approximately 120,000 bases per hour-and a base read length of at least 500 bases per lane. The electrophoresis system and its associated sample loader will minimize the use of sequencing reagents thereby reducing overall reagent costs. The electrophoresis system we propose will be fully integrated with components being developed under other funded efforts. These other complementary projects include the development of a high-speed sample preparation facility and sequence data management and assembly tools. The specific objectives of this proposal are to develop 1) an automated sample loading system capable of loading 384 samples simultaneously and using 10X less DNA than conventional electro-kinetic injection techniques used for capillary electrophoresis systems; 2) arrays of sealed glass microchannels incorporating 384 separate electrophoresis channels and associated input ports and low-noise optical detection chambers; 3) a fluorescence detection system utilizing a CCD detector for the simultaneous detection of four different spectral bands. 4) the necessary electronics and software for real-time data collection; and 5) integrated base-calling software designed to lengthen base calls and capable of producing rigorous, probability-based assessments of sequence quality. The array of microchannels and sample loader will be built using microfabrication technology to integrate 384 separation columns on glass substrate(s). The laser induced fluorescence detection system will be constructed with an argon-ion laser for excitation and a spectrograph and Charge Coupled Device for four color fluorescence detection. The base calling software will incorporate DNA transport models of peak shape, nobility, and diffusion. Incorporating these models will enable the software to deconvolve runs of bases more reliably, as well as assess the quality of the resulting deconvolutions. The work will be completed in three phases: 1) design optimization; 2) unit construction; and 3) system integration and optimization.

描述：（申请人的摘要）我们DNA的2 - 4年目标 测序仪器计划是组装全面的 每24小时至少可以处理和分析至少3000个克隆的设施 测序系统。在此应用程序中，我们建议开发一个 该设施的组成部分 - 高度自动化的电泳系统 使用集成的基础呼叫软件，包括适当的实时 分析工具。完成后，电泳系统将分析 384个DNA样品同时运行的周期时间小于 三个小时。它的峰测序速率大约 每小时120,000个基地和一个基本读取长度至少为500个基地 车道。电泳系统及其相关的样品加载器将 最小化测序试剂的使用，从而减少总体试剂 费用。我们提出的电泳系统将完全集成 在其他资助的努力下开发了组件。这些 补充项目包括开发高速样本 准备设施和序列数据管理和组装工具。 该提议的具体目标是发展 1）能够加载384个样品的自动样品加载系统 同时使用的DNA比常规电动运动少10倍 用于毛细管电泳系统的注射技术； 2）密封的玻璃微通道阵列包含384个单独的 电泳通道和相关输入端口和低噪声光学 检测室； 3）使用CCD检测器的荧光检测系统 同时检测四个不同的光谱带。 4）实时数据收集的必要电子和软件； 和 5）旨在延长基本调用和 能够对序列进行严格的，基于概率的评估 质量。 将使用微通道和样品加载器的数组 微加工技术以整合玻璃上的384个分离柱 底物。激光诱导的荧光检测系统将是 用氩离子激光构建以进行激发和光谱仪和 充电耦合装置，用于四种颜色荧光检测。基地 通话软件将结合峰形的DNA传输模型， 贵族和扩散。结合这些模型将使 更可靠的底座运行软件，并评估 由此产生的反应质量。工作将完成 在三个阶段中：1）设计优化； 2）单位建设； 3） 系统集成和优化。