SBIR-STTR Award

This research involves the development a disposable purification cassette and bench-top electrophoretic processing instrument that will purify one milligram or more of plasmid DNA from recombinant culture in 60 minutes. The purification method to be employed in this work is a derivative of proven electrophoretic separation technology that our company developed for automated mini-prep DNA purification. Data obtained prior to Phase I demonstrated that up to 0.3 milligrams of highly pure plasmid DNA could be automatically purified from 50 mls of liquid bacterial culture loaded directly into a prototype cassette. This preliminary data indicates that cassettes could be made to process up to 150 mls of culture and thus yield 1 mg or more of plasmid DNA. Phase I work will establish the proof-of-concept for the product. Aims of Phase I include: optimization of the cassette design to determine the limitations of the scaled-up process, and optimization of the electrophoretic voltage program to reduce the run time to one hour, without yield loss, and with minimal user steps. The process will also be tested for purification of genomic DNA from bacterial and mammalian cells. A microprocessor control circuit will be constructed in Phase I that will be the basis of the actual processing instrument to be developed in Phase II. The products from this research will be faster and far easier to use than any instrument or manual kit currently available. The disposables of this product are projected to cost $10 per sample, while the bench-top instrument will have a price of ~$2000. The method has essentially no moving parts. This work will greatly reduce the labor and cost required to purify milligram amounts of plasmid DNA. Automated purification of plasmid and genomic DNA participates in a multi- hundred million-dollar nucleic acid purification market. The product has the potential to generate $10-20 million per year in revenue.

Public Health Relevance:
The work will create a bench-top instrument and disposable cassettes that will allow for fully automated purification of milligram quantities of plasmid and genomic DNA. This technology will be significantly less expensive and far less time consumptive that any existing instrument or manual kit available.

Phase II work involves the development and optimization of a bench-topelectrophoretic processing instrument and disposable cassettes that will purify milligramquantities of plasmid DNA from recombinant culture in 90 minutes. Phase I results demonstrated that 1.1 milligrams of plasmid DNA could beautomatically purified from 200 mls of bacterial culture which was loaded directly into thesample well of a prototype cassette. The resulting DNA was highly pure and active as atemplate for automated DNA sequencing. Specific aims of Phase II include: yield optimization by refinement of theelectrophoretic voltage program, optimization of lysis chemistry, refinement of thecassette design, development of the method for concentration of the purified DNA,optimization of the protocol for medium-copy-number and large-size plasmids;determination of transfection activity of purified DNA; and refinement of the method forgenomic DNA purification. Phase II aims for instrument development include construction and testing of theinstrument electronic circuitry and development of the operating software. The electroniccomponents to be assembled and tested include: a microprocessor control board, circuitrelay board, LCD readout, membrane touch switch, power supplies, pump and valveassembly, fluid level switch, and cooling fans; all of which will be controlled by theprogrammable microprocessor board and operating software. Phase II work will be carried out to finalize the cassette design and contract forconstruction of injection molds to create the cassettes and instrument case. Acomprehensive instruction manual and specifications will be written. The bench-top instrument will have a price of ~$3000, while the disposables areprojected to cost $12 per sample. MacConnell Research will be able to directly sell DNApurification cassettes and the instrument developed from this work after Phase II. Theproducts created by this work have the potential to generate $10-20 million per year inrevenue.

Thesaurus Terms:
Bacteria;Chemistry;Computer Software;Contracts;Cost;Cytolysis;Data;Design;Development;Device Or Instrument Development;Dna;Dna Purification;Dna Sequence;Electronics;Genomic Dna;Goals;Improved;Injection Of Therapeutic Agent;Instruction;Instrument;Liquid Substance;Mammalian Cell;Manuals;Marketing;Membrane;Method Development;Methodology;Methods;Microprocessor;Milligram;Molds;Nucleic Acid Purification;Nucleic Acids;Phase;Plasmid Dna;Plasmids;Power Sources;Preparation;Price;Process;Programs;Protocols Documentation;Prototype;Public Health Relevance;Pump;Recombinants;Research;Sampling;Technology;Testing;Time;Touch Sensation;Transfection;Voltage;Work;Writing;