Automated Large and Mid Scale Nucleic Acid Preparation

点击翻译按钮获取中文摘要

Phase II work involves the development and optimization of a bench-top electrophoretic processing instrument and disposable cassettes that will purify milligram quantities of plasmid DNA from recombinant culture in 90 minutes. Phase I results demonstrated that 1.1 milligrams of plasmid DNA could be automatically purified from 200 mls of bacterial culture which was loaded directly into the sample well of a prototype cassette. The resulting DNA was highly pure and active as a template for automated DNA sequencing. Specific aims of Phase II include: yield optimization by refinement of the electrophoretic voltage program, optimization of lysis chemistry, refinement of the cassette 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 for genomic DNA purification. Phase II aims for instrument development include construction and testing of the instrument electronic circuitry and development of the operating software. The electronic components to be assembled and tested include: a microprocessor control board, circuit relay board, LCD readout, membrane touch switch, power supplies, pump and valve assembly, fluid level switch, and cooling fans; all of which will be controlled by the programmable microprocessor board and operating software. Phase II work will be carried out to finalize the cassette design and contract for construction of injection molds to create the cassettes and instrument case. A comprehensive instruction manual and specifications will be written. The bench-top instrument will have a price of ~$3000, while the disposables are projected to cost $12 per sample. MacConnell Research will be able to directly sell DNA purification cassettes and the instrument developed from this work after Phase II. The products created by this work have the potential to generate $10-20 million per year in revenue.