Rapid, Universal, Low Cost Automated Genomic DNA Purification from Micro Samples

描述(申请人提供)：这项工作的目的是开发一种低成本、自动化、多样本的设备和方法，允许快速有效地从各种组织、细胞和细菌样本中纯化基因组DNA。这一过程将被优化，以从含有从微克到微克的核酸的样品中提纯DNA。该方法使用正向和反向电泳法从样品处理盒中的总细胞裂解液中分离基因组DNA，不需要移动部件，可以在不到20分钟的时间内完成。该方法可以并行处理样品，并且可以被微型化，以便一次可以同时提纯几个或数百个样品。用第一阶段之前构建的原型盒进行的试验证明，用这种方法制备的DNA纯度很高，可以直接用于PCR扩增和其他分子生物学应用。这种基因组DNA纯化方法结合了我公司为自动纯化质粒DNA而开发的技术，以及我们开发的无需液体缓冲的琼脂糖凝胶运行技术。新的纯化方法与我们公司目前的仪器技术不同，整个过程可以在盒的起始井中进行，即使样品中有微量核酸，也可以进行几乎定量的回收。盒式磁带处理仪由一个简单、廉价的装置组成，只提供电源和用于净化的微处理器控制电路。我们在第一阶段的目标是证明该方法可以普遍应用于从各种大小的样本中提纯细菌、哺乳动物和植物的DNA。初步数据显示，该方法可以从仅含毫微克DNA的样品中提纯DNA。使用这项技术的最终产品预计每个样品的成本不到0.35美元，其中处理设备的成本约为1000美元。这些标准与目前的DNA纯化产品相比是有利的，手动试剂盒的每份准备费用通常为1美元，自动化仪器的每份准备费用为10,000-60,000美元。因为我们的新盒式磁带没有移动部件，所以它可以用每个盒式磁带的高密度样本通道来建造，例如在其4.5“x 2”x 1“尺寸内有24或48个通道。由此产生的产品将显著更易于操作，并且需要的工作台空间比目前可用的任何手动或自动仪器都要少。在第一阶段，将进行实验以改进纯化方法的性能，包括优化步骤：允许最短运行时间的盒的物理结构、起始样品的裂解条件以及用于分离DNA的电压施加程序。将进行试验，以测试从血液和细菌中递减提纯DNA的工艺，以确定该方法的产量下限。这一过程将用酵母细胞、植物组织和几个细菌菌株进行测试；我们将测试一种用饱和缓冲液的灯芯代替运行缓冲液的方法。最后，我们将对执行程序运行的电源/仪器的设计进行概念化，以使该组件尽可能简单和经济。一种新的仪器和装置将被开发出来，它将彻底改变从细胞、细菌、血液和植物组织中提取基因组DNA的自动化。这项技术将通过提供更便宜、更快、更好的解决方案来提纯基因组DNA，从而提高国家在疾病基础研究和临床应用方面的能力。

DESCRIPTION (provided by applicant): The aim of this work is to develop a low cost, automated, multi-sample device and method that will allow for rapid and efficient purification of genomic DNA from a wide variety of tissue, cell and bacterial samples. This process will be optimized to purify DNA from samples that contain nucleic acid ranging from picograms to micrograms. The method uses forward and reverse electrophoretic separation of genomic DNA from total cell lysate in a sample-processing cassette that requires no moving parts, and can be performed in less than 20 minutes. The method can process the samples in parallel and can be miniaturized to allow simultaneous purification of a few, or hundreds of samples at one time. Trials with prototype cassettes constructed prior to Phase I have proven that DNA prepared by this method is highly pure, and can be used directly in PCR amplification and other molecular biology applications. This genomic DNA purification method combines technology that our company developed for automated plasmid DNA purification with technology we developed for running agarose gels without liquid buffer. The new purification methodology differs from our company's current instrument technology in that the entire process can be carried out in the starting well of the cassette, which allows almost quantitative recovery even with trace amounts of nucleic acid in the sample. The processing instrument for the cassette consists of a simple, inexpensive device providing only the power supply and microprocessor control circuit for the purification. Our goal in Phase I is to demonstrate that the method can be universally applied to the purification of bacterial, mammalian, and plant DNA from a wide range of sample sizes. Preliminary data shows that the method can purify DNA from samples containing only nanograms of DNA. The final product using this technology is predicted to cost less than $0.35 per sample with a processing device that will cost approximately $1000. These criteria compare favorably to current DNA purification products that are typically $1 per prep for manual kits and $10,000 - $60,000 for automated instruments. Because our new cassette has no moving parts, it can be constructed with a high density of sample lanes per cassette, such as 24 or 48 lanes within its 4.5" x 2" x 1" dimensions. The resulting product will be significantly easier to operate, and require less bench space than any manual or automated instrument currently available. In Phase I, experiments will be carried out to improve the performance of the purification method, including steps to optimize: the physical structure of the cassette to allow the shortest run time, the conditions of lysis of the starting sample, and the voltage application program for separation of the DNA. Trials will be carried out to test the process for purification of DNA from blood and bacteria in decreasing amounts to establish the lower yield limits of the method. The process will be tested with yeast cells, plant tissue, and several bacterial strains; and we will test a method of substituting buffer-saturated wicks for the running buffer. Finally, we will conceptualize the design of the power supply/instrument that will perform the programmed run in an effort to make this component as simple and cost effective as possible. A novel instrument and device will be developed that will revolutionize the automation of genomic DNA from cell, bacteria, blood, and plant tissue. The technology will advance the Nations capability in basic research of disease and clinical applications by providing a cheaper, faster, better solution to the purification of genomic DNA.