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

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 p rocess 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 l oading 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 reproducibil ity 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 c ontaining 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 seq uencing. (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 pr ep, 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 gen omic 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 Pha se 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