Automated Large and Mid Scale Nucleic Acid Preparation

描述(申请人提供)：第二阶段工作包括开发和优化台式电泳仪和一次性盒式磁带，将在90分钟内从重组培养物中纯化毫克量的质粒DNA。第一阶段的结果表明，可以从200mls的细菌培养物中自动提纯1.1毫克的质粒DNA，这些细菌培养物直接加载到样盒的样品井中。得到的DNA纯度高，活性高，可作为DNA自动测序的模板。第二阶段的具体目标包括：通过优化电泳电压程序来优化产量，优化裂解化学，优化盒设计，开发纯化DNA的浓缩方法，优化中等拷贝数和大小的质粒的操作规程，测定纯化DNA的转染活性，以及改进基因组DNA纯化方法。第二阶段的目标是开发仪器，包括仪器电子电路的建造和测试以及操作软件的开发。待组装和测试的电子元件包括：微处理器控制板、电路中继板、LCD读取器、薄膜触摸开关、电源、泵和阀门组件、液位开关和冷却风扇；所有这些都将由可编程微处理器板和操作软件控制。将进行第二阶段的工作，以最后确定盒式磁带的设计和建造注射模具的合同，以制造盒式磁带和仪表盒。将编写一份全面的说明手册和规范。台式仪器的价格约为3000美元，而一次性仪器预计每个样品的价格为12美元。在第二阶段之后，麦康奈尔研究公司将能够直接销售DNA提纯盒式磁带和由这项工作开发的仪器。这项工作创造的产品每年有可能产生1000-2000万美元的收入。

DESCRIPTION (provided by applicant): 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.