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

从微量样品中快速、通用、低成本自动化基因组 DNA 纯化

• 批准号: 7324867
• 负责人: WILLIAM P MACCONNELL
• 金额: $ 10万
• 依托单位: MACCONNELL RESEARCH CORPORATION
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7324867
• 关键词: Automation; Bacteria; Basic Science; Blood; Buffers; Cells; Cells Plant Tissue; Chemistry; Condition; Cytolysis; DNA; DNA purification; Data; Devices; Dimensions; Disease; Doctor of Philosophy; Gel; Genomics; Goals; Injection of therapeutic agent; Laboratories; Left; Liquid substance; Manuals; Methodology; Methods; Microprocessor; Molds; Molecular Biology, Other; Nucleic Acids; Performance; Phase; Plant DNA; Plants; Polymerase Chain Reaction; Power Sources; Preparation; Price; Procedures; Process; Purpose; Range; Recovery; Running; Sample Size; Sampling; Sepharose; Side; Solutions; Structure; Technology; Testing; Time; Tissues; Variant; Work; Yeasts; clinical application; concept; cost; cost effective; density; design; improved; instrument; miniaturize; novel; plasmid DNA; programs; prototype; research study; voltage

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.

描述（由申请人提供）：这项工作的目的是开发一种低成本、自动化、多样品装置和方法，其将允许从各种组织、细胞和细菌样品中快速有效地纯化基因组DNA。这一过程将被优化，以从含有皮克至微克核酸的样品中纯化DNA。该方法使用正向和反向电泳分离基因组DNA从总细胞裂解物中的样品处理盒，不需要移动部件，可以在不到20分钟内进行。该方法可以并行处理样品，并且可以小型化以允许同时纯化几个或数百个样品。在I期之前构建的原型盒的试验已经证明，通过该方法制备的DNA是高纯度的，并且可以直接用于PCR扩增和其他分子生物学应用。这种基因组DNA纯化方法结合了我们公司开发的用于自动质粒DNA纯化的技术和我们开发的用于在没有液体缓冲液的情况下运行琼脂糖凝胶的技术。新的纯化方法与我们公司目前的仪器技术不同之处在于，整个过程可以在试剂卡的起始孔中进行，即使样品中含有微量核酸，也可以进行几乎定量的回收。用于盒的处理仪器由简单、廉价的装置组成，该装置仅提供用于纯化的电源和微处理器控制电路。我们在第一阶段的目标是证明该方法可以普遍适用于从广泛的样品量中纯化细菌，哺乳动物和植物DNA。初步数据显示，该方法可以从仅含有纳克DNA的样品中纯化DNA。使用该技术的最终产品预计每个样品的成本低于0.35美元，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，目前的DNA纯化产品通常是手动试剂盒每次制备1美元，自动仪器每次制备10，000 - 60，000美元。因为我们的新盒没有移动部件，所以它可以被构造成每个盒具有高密度的样品泳道，例如在其4.5”x 2”x 1”尺寸内具有24或48个泳道。由此产生的产品将更容易操作，并且比目前可用的任何手动或自动仪器需要更少的工作台空间。在第一阶段，将进行实验以改善纯化方法的性能，包括优化步骤：盒的物理结构以允许最短的运行时间，起始样品的裂解条件，以及用于分离DNA的电压应用程序。将进行试验以测试从血液和细菌中纯化DNA的过程，以减少数量，以确定该方法的产率下限。这个过程将用酵母细胞、植物组织和几种细菌菌株进行测试;我们将测试一种用缓冲液饱和的吸液芯代替运行缓冲液的方法。最后，我们将对执行编程运行的电源/仪器的设计进行概念化，以使该组件尽可能简单且具有成本效益。将开发一种新的仪器和设备，这将彻底改变从细胞、细菌、血液和植物组织中提取基因组DNA的自动化。该技术将通过提供更便宜、更快、更好的基因组DNA纯化解决方案，提高国家在疾病基础研究和临床应用方面的能力。