Rapid, Very Low Cost, Automated DNA Purification Device

描述（由申请人提供）：这项工作将创造创新的产品，允许快速，低成本，自动化，多样品基因组DNA纯化从许多样品类型和浓度。我们将在第一阶段进一步开发和验证的新技术，使用一次性盒装置和低成本处理支架，通过独立的半干电泳方法自动纯化DNA。Cascadium可以被构建为包含多个泳道，其允许在一次运行中同时纯化几个或多达96个样品。该方法不需要移动部件，可以在不到10分钟内完成。由于其简单性，盒的预计成本将为每个样本0.4 - 0.9美元，而便携式处理支架的成本将低于1-12样本版本的100美元，或96样本版本的250美元。该产品如果完全实现，将彻底改变临床或参考实验室诊断程序的DNA制备方式。 在此提议之前，利用纯化盒和处理装置的原型进行的工作表明，从血液、唾液或组织中制备的DNA是高纯度的，可以直接用于PCR扩增以及其他分子生物学应用。初步工作还表明，该方法可以从宽范围的样品浓度开始开始，因为起始样品可以稀释100-250倍，并且该过程仍然产生活性DNA模板。卡匣的最终版本将是可回收的。 基因组DNA纯化方法利用了我们公司开发的用于自动质粒DNA纯化的技术变体，结合我们发明的用于在没有运行缓冲液的情况下运行琼脂糖凝胶的技术（24，26）。该方法允许在一次性盒的起始孔中进行整个纯化过程。基因组DNA在其纯化过程中实际上不离开加载孔的事实允许接近定量的回收，即使是痕量的核酸。第一阶段的工作将建立产品的概念验证。第一阶段的具体目标是：（1）进一步开发和改进原型DNA纯化盒，使运行时间减少到10分钟或更短。（2）进一步测试减少样品量和优化裂解条件的方法，以确定其适用的哺乳动物和细菌样品范围。(3)开发加工支架的电路和组件，并准确估算其制造成本。(4)在各种核酸诊断程序中测定所得DNA的活性，如RT-PCR和下一代测序。 公共卫生相关性：该提案将创造创新产品，允许从许多样品类型和浓度中快速、低成本、自动化、多样品基因组DNA纯化。该新技术使用一次性的、可回收的盒式装置和低成本的处理支架，通过电泳手段自动纯化DNA，而无需运行缓冲液浴或铂电极。

DESCRIPTION (provided by applicant): This work will create innovative products that allow rapid, low cost, automated, multi- sample genomic DNA purification from many sample types and concentrations. The novel technology that we will further develop and validate in Phase I, uses disposable cassette device and low cost processing cradle to automatically purify DNA by self-contained, semi-dry electrophoretic means. Cassettes can be constructed to contain multiple lanes that allow simultaneous purification of a few, or up to 96 samples in one run. The method requires no moving parts and can be performed in less than 10 minutes. The projected cost of cassettes, due to their simplicity, will be $0.4 - $0.9 per sample, while the portable processing cradle will cost less than $100 for a 1-12 sample version, or $250 for a 96-sample version. This product, if fully realized, will revolutionize the way that DNA is prepared for clinical or reference laboratory diagnostic procedures. Work performed prior to this proposal with prototypes of the purification cassette and processing device showed that DNA prepared from blood, saliva or tissue is highly pure and can be used directly in PCR amplification, as well as other molecular biology applications. Preliminary work also showed that the method could begin with a wide range of sample concentrations, as the starting sample could be diluted 100-250 fold and process still yielded active DNA template. The final version of the cassettes will be recyclable. The genomic DNA purification method utilizes a variation of technology that our company developed for automated plasmid DNA purification, combined with technology that we invented for running agarose gels without running buffer (24,26). The method allows the entire purification process to be carried out in the starting well of the disposable cassette. The fact that the genomic DNA does not actually leave the loading well during its purification allows for near quantitative recovery, even with trace amounts of nucleic acid. Phase I work will establish the proof-of-concept for the product. Specific aims of Phase I are to: (1) further develop and refine the prototype DNA purification cassette to allow the run time to be reduced to 10 minutes or less. (2) Further test the method with reduced sample sizes and with optimized lysis conditions to determine the range of mammalian and bacterial samples for which it is applicable. (3) Develop the circuitry and components of the processing cradle and accurately estimate its manufacturing cost. (4) Determine the activity of the resulting DNA in a variety of nucleic acid diagnostic procedures such as RT-PCR and next generation sequencing. PUBLIC HEALTH RELEVANCE: This proposal will create innovative products that allow rapid, low cost, automated, multi-sample genomic DNA purification from many sample types and concentrations. The novel technology uses disposable, recyclable cassette device and low cost processing cradle to automatically purify DNA by electrophoretic means, without running buffer baths or platinum electrodes.