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，可以快速高效地从各种组织、细胞和细菌样本中纯化基因组DNA。这一过程将进行优化，以处理含有从皮克到微克范围内的核酸的样本。这种新的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，样品处理盒在样品装载井的两边都有琼脂糖片段。盒可以被构造为包含多个通道，从而允许在编程的电泳分离运行期间同时处理多个样品。样品通道和装载井可以被微型化，以允许一次同时提纯几个或数百个样品。该方法不需要移动部件，可以在不到15分钟的时间内完成。用之前和第一阶段构建的原型盒进行的试验表明，用这种方法制备的DNA纯度很高，可以用于PCR扩增和其他分子生物学应用。我们的第二阶段目标是证明该电泳过程可以普遍和重复性地应用于从各种大小的样品中提纯细菌、哺乳动物和植物的DNA。I期数据显示，该方法可以从仅含毫微克基因组DNA的样品中提纯DNA。在第二阶段，将进行实验，以改进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)改进分离过程，以接受含有微量DNA的较小样本量。(3)扩大细菌、植物、哺乳动物细胞和酵母的样品种类；(4)进一步改进裂解化学和前处理条件。(5)用RFLP和PCRDNA测序检测纯化DNA的活性。(6)开发和测试缓冲芯和预制的一次性芯盒。(7)制定从少量全血或干血和口腔拭子中提纯DNA的方案。(8)开发一种可提供高达300伏电压的改进版本的直流电源。(9)修改仪器操作软件，完成基因组DNA的纯化。(10)为开发的产品编写说明书和说明书。使用这项技术的最终产品，每个样品的成本不到0.50美元，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纯化试剂盒相比是有利的，目前DNA纯化试剂盒的价格通常为每次1美元，而用于这一目的的自动化仪器的价格为20,000-80,000美元。因为这种新的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构造，例如在其4.5“x 2”x 1“尺寸内有24或48个通道。与目前可用的任何手动或仪表化产品相比，该产品的操作也更加容易，需要的工作台空间也更少。它将为基因组DNA准备的自动化问题提供更便宜、更快和更好的解决方案，并将被设计成适合小型实验室的需求。这项工作将产生的产品将面向全球40,000多个分子生物学研究实验室，潜在市场价值为3500万美元。在第二阶段结束时，麦康奈尔研究公司将能够开始制造和销售从这项工作中开发出来的仪器和磁带。与公共卫生相关：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，以便快速有效地从各种组织、细胞和细菌样本中提纯基因组DNA。这一过程将进行优化，以处理含有从皮克到微克范围内的核酸的样本。

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.