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

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，多样本的设备和方法，可以快速有效地从各种组织，细胞和细菌样本中纯化基因组DNA。该过程将被优化，以处理含有从皮克到微克范围内核酸的样品。这种新方法在样品处理盒中使用正向和反向电泳分离基因组DNA，从总细胞裂解液中分离出琼脂糖，该样品处理盒在样品装载孔的两侧都有琼脂糖。盒式磁带可以构造成包含多个通道，允许多个样品在编程电泳分离运行期间同时处理。样品通道和装载孔可以小型化，以允许同时纯化几个或数百个样品。该方法不需要任何活动部件，可在15分钟内完成。在第一阶段之前和期间构建的原型盒试验表明，通过该程序制备的DNA纯度很高，可用于PCR扩增和其他分子生物学应用。我们第二阶段的目标是证明电泳过程可以普遍和可重复性地应用于细菌，哺乳动物和植物DNA的纯化，从广泛的样本量。一期数据显示，该方法可以从仅含纳克基因组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，以实现最短的纯化时间；(2)改进分离过程，以接受含有微量DNA的更小样本量。(3)扩大细菌、植物、哺乳动物细胞、酵母的样品种类清单；(4)进一步完善裂解化学和预处理条件。(5)利用RFLP和PCR测序检测纯化DNA的活性。(6)开发和测试缓冲灯芯和预先制造的一次性灯芯盒。(7)制定从小体积全血或干血和口腔拭子中纯化DNA的方案。(8)开发一种可提供高达300伏直流电源的改进版本。(9)修改仪器操作软件，完成基因组DNA纯化。（10）编写所开发产品的使用说明书和规格书。使用该技术的最终产品每个样品的成本低于0.50美元，其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（每次准备通常为1美元）和用于此目的的自动化仪器（价格为2万至8万美元）相比具有优势。由于这种新卡带没有活动部件，因此可以在每个卡带中构建高密度的样品通道，例如在4.5“x 2”x 1”尺寸内的24或48个通道。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器仪表产品目前可用。它将为自动化基因组DNA制备问题提供更便宜、更快和更好的解决方案，并将被设计为适合小型实验室的需要。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，潜在市场为3500万美元。在第二阶段结束时，麦康奈尔研究中心将能够开始生产和销售从这项工作中开发出来的仪器和磁带。公共卫生相关性：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，以便从各种组织、细胞和细菌样本中快速有效地纯化基因组DNA。该过程将被优化，以处理含有从皮克到微克范围内核酸的样品。

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.