Automated Large and Mid Scale Nucleic Acid Preparation

自动化大中型核酸制备

• 批准号: 8648447
• 负责人: WILLIAM P MACCONNELL
• 金额: $ 49.25万
• 依托单位: MACCONNELL RESEARCH CORPORATION
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-03-01 至 2016-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8648447
• 关键词: Bacteria; Chemistry; Computer software; Contracts; Cytolysis; DNA; DNA Sequence; DNA purification; Data; Development; Device or Instrument Development; Electronics; Genomic DNA; Goals; Injection of therapeutic agent; Instruction; Liquid substance; Mammalian Cell; Manuals; Marketing; Membrane; Methodology; Methods; Microprocessor; Molds; Nucleic Acids; Phase; Plasmids; Power Sources; Preparation; Price; Process; Protocols documentation; Pump; Recombinants; Research; Sampling; Technology; Testing; Time; Touch sensation; Transfection; Work; Writing; cost; design; improved; instrument; method development; milligram; nucleic acid purification; plasmid DNA; programs; prototype; public health relevance; voltage

Phase II work involves the development and optimization of a bench-top electrophoretic processing instrument and disposable cassettes that will purify milligram quantities of plasmid DNA from recombinant culture in 90 minutes. Phase I results demonstrated that 1.1 milligrams of plasmid DNA could be automatically purified from 200 mls of bacterial culture which was loaded directly into the sample well of a prototype cassette. The resulting DNA was highly pure and active as a template for automated DNA sequencing. Specific aims of Phase II include: yield optimization by refinement of the electrophoretic voltage program, optimization of lysis chemistry, refinement of the cassette design, development of the method for concentration of the purified DNA, optimization of the protocol for medium-copy-number and large-size plasmids; determination of transfection activity of purified DNA; and refinement of the method for genomic DNA purification. Phase II aims for instrument development include construction and testing of the instrument electronic circuitry and development of the operating software. The electronic components to be assembled and tested include: a microprocessor control board, circuit relay board, LCD readout, membrane touch switch, power supplies, pump and valve assembly, fluid level switch, and cooling fans; all of which will be controlled by the programmable microprocessor board and operating software. Phase II work will be carried out to finalize the cassette design and contract for construction of injection molds to create the cassettes and instrument case. A comprehensive instruction manual and specifications will be written. The bench-top instrument will have a price of ~$3000, while the disposables are projected to cost $12 per sample. MacConnell Research will be able to directly sell DNA purification cassettes and the instrument developed from this work after Phase II. The products created by this work have the potential to generate $10-20 million per year in revenue.

第二阶段的工作包括开发和优化工作台 将净化毫克的电泳仪和一次性盒式磁带 在90分钟内从重组培养物中提取大量的质粒DNA。 I期结果显示，1.1毫克的质粒DNA可以 从200毫升的细菌培养物中自动提纯，直接加载到 原型盒的样品井。得到的DNA纯度很高，作为一种 用于自动DNA测序的模板。 第二阶段的具体目标包括：通过完善 电泳电压程序，裂解化学的优化，精制 盒设计，纯化DNA浓缩方法的发展， 中拷贝数和大小质粒检测方法的优化 纯化的DNA的转染活性的测定和方法的改进 基因组DNA提纯。 第二阶段的目标是开发仪器，包括建造和测试 仪器电子电路及操作软件的开发。电子产品 待组装和测试的组件包括：微处理器控制板、电路 中继板、LCD读取器、薄膜触摸开关、电源、泵和阀 组件、液位开关和冷却风扇；所有这些都将由 可编程微处理器板卡和操作软件。 将进行第二阶段工作，以最后确定盒式磁带的设计和合同 制造盒式磁带和仪表盒的注塑模具。一个 将编写全面的说明手册和规范。 台式仪器的价格约为3000美元，而一次性仪器的价格为 预计每个样品的价格为12美元。麦康奈尔研究公司将能够直接出售DNA 净化盒和第二阶段后在此工作基础上开发的仪器。 这项工作创造的产品每年有可能产生1000-2000万美元的 收入。