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: 7672246
• 负责人: WILLIAM P MACCONNELL
• 金额: $ 37.88万
• 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 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7672246
• 关键词: Address; Applications Grants; Bacteria; Biological Assay; Blood; Buffers; Cells; Chemistry; Computer software; Cytolysis; DNA; DNA Sequence; DNA purification; Data; Devices; Dimensions; Doctor of Philosophy; Gel; Genomics; Goals; Instruction; Laboratories; Laboratory Research; Left; Mammalian Cell; Manuals; Marketing; Methodology; Methods; Molecular Biology; Nucleic Acids; Performance; Phase; Plant DNA; Plants; Power Sources; Preparation; Price; Procedures; Process; Protocols documentation; Recovery; Reproducibility; Research; Restriction fragment length polymorphism; Running; Sample Size; Sampling; Sepharose; Side; Small Business Innovation Research Grant; Solutions; Swab; Technology; Testing; Time; Tissues; Variant; Whole Blood; Work; Writing; Yeasts; cost; density; design; improved; instrument; miniaturize; novel; plasmid DNA; programs; prototype; public health relevance; research study

DESCRIPTION (provided by applicant): The aim of the work is to develop a low cost, automated, multi-sample device and method that will allow rapid and efficient purification of genomic DNA from a wide variety of tissue, cell, and bacterial samples. The process will be optimized to work with samples that contain nucleic acid in a range from picograms to micrograms. The novel method uses forward and reverse electrophoretic separation of genomic DNA from total cell lysate in a sampleprocessing cassette that has sections of agarose cast on either side of sample loading wells. Cassettes can be constructed to contain multiple lanes, allowing multiple samples to be processed simultaneously during a programmed electrophoretic separation run. The sample lanes and loading wells can be miniaturized to allow simultaneous purification of a few, or hundreds of samples at one time. The method requires no moving parts and can be performed in less than 15 minutes. Trials with prototype cassettes constructed prior and during phase I, have shown that DNA prepared by this procedure is highly pure, and can be used in PCR amplification and other molecular biology applications. Our Phase II goal is to demonstrate that the electrophoretic process can be universally and reproducibility applied to the purification of bacterial, mammalian, and plant DNA from a wide range of sample sizes. Phase I data showed that the method can purify DNA from samples containing only nanograms of genomic DNA. In Phase II, experiments will be carried out to improve the performance method developed prior to and during phase I, including steps to: (1) Further optimize the electrophoretic run programs to allow the shortest purification time (2) Refine the separation process to accept smaller sample volumes containing trace amounts of DNA. (3) Expand the list of sample types of bacteria, plant, mammalian cells, and yeast, (4) Further improve the lysis chemistry and pre-treatment conditions. (5) Assay the activity of the purified DNA using RFLP, and PCR DNA sequencing. (6) Develop and test buffer wicks and pre-manufactured disposable wick-cassettes. (7) Develop a protocol for DNA purification from small volumes of whole blood or dried blood and buccal swabs. (8) Develop an improved version of the DC power supply that will deliver up to 300 volts. (9) Modify the operating software of the instrument to accomplish the genomic DNA purification. (10) Write an instruction manual and specifications for the developed product. The final product using this technology will cost less than $0.50 per sample with a processing device that is a relatively simple adaptation of the Mini-Prep 96 instruments that our company currently sells. These criteria compare favorably to current DNA purification kits that are typically $1 per prep, and to automated instruments for this purpose, which are priced at $20,000 - $80,000. Because this 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. This product will also be significantly easier to operate and require less bench space than any manual or instrumented product currently available. It will provide a less expensive, faster, and better solution to the problem of automating genomic DNA preparations, and will be designed to fit the needs of the small laboratory. The products that will result from this work will address the 40,000+ molecular biology research labs worldwide, with a potential market of $35 million. At the end of Phase II, MacConnell Research will be able to begin manufacturing and selling the instruments and cassettes developed from this work. PUBLIC HEALTH RELEVANCE: The aim of the work is to develop a low cost, automated, multi-sample device and method that will allow rapid and efficient purification of genomic DNA from a wide variety of tissue, cell, and bacterial samples. The process will be optimized to work with samples that contain nucleic acid in a range from picograms to micrograms.

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，多样品的设备和方法，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。该过程将进行优化，以处理含有皮克至微克范围内核酸的样品。该新方法使用正向和反向电泳从样品处理盒中的总细胞裂解物中分离基因组DNA，该样品处理盒在样品加载威尔斯孔的两侧具有琼脂糖浇铸部分。Cascadion可以被构造成包含多个泳道，允许在编程的电泳分离运行期间同时处理多个样品。样品泳道和上样威尔斯孔可以小型化，以允许同时纯化几个或数百个样品。该方法不需要移动部件，可以在15分钟内完成。在第一阶段之前和期间用构建的原型盒进行的试验表明，通过该方法制备的DNA是高纯度的，并且可用于PCR扩增和其他分子生物学应用。我们的第二阶段的目标是证明，电泳过程可以普遍和再现性适用于从广泛的样品量的细菌，哺乳动物和植物DNA的纯化。第一阶段的数据表明，该方法可以从仅含有纳克基因组DNA的样品中纯化DNA。在第二阶段，将进行实验，以改善在第一阶段之前和期间开发的性能方法，包括以下步骤：（1）进一步优化电泳运行程序，以允许最短的纯化时间（2）优化分离过程，以接受含有微量DNA的较小样品体积。(3)扩大细菌、植物、哺乳动物细胞和酵母的样品类型列表。（4）进一步改进裂解化学和预处理条件。(5)用限制性片段长度多态性（RFLP）和聚合酶链反应（PCR）DNA测序法检测纯化DNA的活性。(6)开发和测试缓冲液芯和预制一次性芯盒。(7)制定从少量全血或干血和口腔拭子中纯化DNA的方案。(8)开发一种改进型直流电源，可提供高达300伏的电压。(9)修改仪器操作软件，完成基因组DNA纯化。(10)为开发的产品编写说明书和规格。使用这种技术的最终产品将花费不到0.50美元，每个样品的处理设备是我们公司目前销售的Mini-Prep 96仪器的相对简单的改编。这些标准与目前的DNA纯化试剂盒（通常每次制备1美元）以及用于此目的的自动化仪器（价格为20，000 - 80，000美元）相比是有利的。因为这种新的盒没有移动部件，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。该产品也将更容易操作，需要更少的工作台空间比任何手动或仪表产品目前可用。它将为自动化基因组DNA制备的问题提供一个更便宜、更快、更好的解决方案，并将被设计为满足小型实验室的需求。这项工作产生的产品将面向全球40，000多个分子生物学研究实验室，潜在市场为3500万美元。在第二阶段结束时，MacConnell Research将能够开始制造和销售从这项工作中开发的仪器和卡匣。 公共卫生关系：这项工作的目的是开发一种低成本，自动化，多样品的设备和方法，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。该过程将进行优化，以处理含有皮克至微克范围内核酸的样品。