SBIR-STTR Award

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

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.

Public Health Relevance:
This Public Health Relevance is not available.

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