This work will create innovative products that allow rapid, low cost, automated, multi- sample genomic DNA purification from many sample types and concentrations. The novel technology that we will further develop and validate in Phase I, uses disposable cassette device and low cost processing cradle to automatically purify DNA by self-contained, semi-dry electrophoretic means. Cassettes can be constructed to contain multiple lanes that allow simultaneous purification of a few, or up to 96 samples in one run. The method requires no moving parts and can be performed in less than 10 minutes. The projected cost of cassettes, due to their simplicity, will be $0.4 - $0.9 per sample, while the portable processing cradle will cost less than $100 for a 1-12 sample version, or $250 for a 96-sample version. This product, if fully realized, will revolutionize the way that DNA is prepared for clinical or reference laboratory diagnostic procedures. Work performed prior to this proposal with prototypes of the purification cassette and processing device showed that DNA prepared from blood, saliva or tissue is highly pure and can be used directly in PCR amplification, as well as other molecular biology applications. Preliminary work also showed that the method could begin with a wide range of sample concentrations, as the starting sample could be diluted 100-250 fold and process still yielded active DNA template. The final version of the cassettes will be recyclable. The genomic DNA purification method utilizes a variation of technology that our company developed for automated plasmid DNA purification, combined with technology that we invented for running agarose gels without running buffer (24,26). The method allows the entire purification process to be carried out in the starting well of the disposable cassette. The fact that the genomic DNA does not actually leave the loading well during its purification allows for near quantitative recovery, even with trace amounts of nucleic acid. Phase I work will establish the proof-of-concept for the product.
Specific aims of Phase I are to: (1) further develop and refine the prototype DNA purification cassette to allow the run time to be reduced to 10 minutes or less. (2) Further test the method with reduced sample sizes and with optimized lysis conditions to determine the range of mammalian and bacterial samples for which it is applicable. (3) Develop the circuitry and components of the processing cradle and accurately estimate its manufacturing cost. (4) Determine the activity of the resulting DNA in a variety of nucleic acid diagnostic procedures such as RT-PCR and next generation sequencing.
This proposal will create innovative products that allow rapid, low cost, automated, multi-sample genomic DNA purification from many sample types and concentrations. The novel technology uses disposable, recyclable cassette device and low cost processing cradle to automatically purify DNA by electrophoretic means, without running buffer baths or platinum electrodes.
