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

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.