Automated Large and Mid Scale Nucleic Acid Preparation

描述（由申请人提供）：II期工作涉及台式电泳处理仪器和一次性试剂卡的开发和优化，这些仪器和试剂卡将在90分钟内从重组培养物中纯化毫克量的质粒DNA。 阶段I的结果表明，可以从200毫升细菌培养物中自动纯化1.1毫克质粒DNA，所述细菌培养物直接装载到原型盒的样品孔中。得到的DNA是高纯度的，并且作为自动化DNA测序的模板具有活性。 第II阶段的具体目标包括：通过优化电泳电压程序优化产量、优化裂解化学、优化盒设计、开发纯化DNA浓缩方法、优化中等拷贝数和大尺寸质粒的方案;测定纯化DNA的转染活性;以及优化基因组DNA纯化方法。 第二阶段的目标是仪器的发展，包括建设和测试仪器的电子电路和操作软件的开发。待组装和测试的电子组件包括：微处理器控制板、电路继电器板、LCD读出器、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和操作软件控制。 将开展第二阶段工作，以最终确定卡匣设计和注塑模具建造合同，以创建卡匣和仪器箱。将编写全面的说明手册和规格。 台式仪器的价格约为3000美元，而一次性仪器预计每个样本的价格为12美元。MacConnell Research将能够在第二阶段之后直接销售DNA纯化盒和从这项工作中开发的仪器。这项工作创造的产品有可能每年产生1000万至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.