Tuyen Nguyen, Randy F. Sivila, Joshua P. Dyer, and William P. MacConnell
MacConnell Research Corporation, San Diego, California
MacConnell Research currently manufactures and sells a low cost automated bench-top instrument that can purify up to 24 samples of plasmid DNA simultaneously in one hour at a cost of $0.65 per sample and under $8000 for the instrument. The patented instrument uses a form of agarose gel electrophoresis to purify the plasmid DNA and electroelutes in into approximately a 20 µl volume. The instrument has many advantages of other robotic and manual methods including that fact that it is two times faster, at least six times less expensive, much smaller in size, easier to operate, less cost per sample, and results in DNA pure enough for direct use in fluorescent automated sequencing. The instrument process begins with bacterial culture which is loaded directly into a disposable cassette in the machine.
In Phase II work we are developing an instrument which simultaneously purifies plasmid DNA from up to 192 (2 X 96) bacterial samples in 1.5 hours. Prototype of this instrument thus far constructed have allowed the purification of 3-7 micrograms of high purity plasmid DNA per lane from 1.5 ml of bacterial culture. We have attempted to optimize all of the: instrument electrophoretic run parameters, lysis chemistry, lysis reagent delivery devices, reagent storage at room temperature, desalting processes and overall instrument mechanical and electronic control. Instrument prototypes have also been used to prepare cosmid or yeast DNA in quantities of 1-5 micrograms per cassette lane. Trials thus far have yielded plasmid DNA of sufficient purity for direct use in automated fluorescent and manual sequencing as well as other molecular biology protocols. We have studied the purity of the resulting DNA when directly sequenced on a Licor 4000 Long Reader and ABI 373A automated DNA sequencers. Results from the Licor 4000 instrument gives routine read lengths of >850 base pairs with 98% accuracy while ABI 373A reads generally exceed 400 base pairs with the similar accuracy.
The proposed 2 X 96-channel instrument will purify up to 1200 plasmid DNA preps per eight hour day. It will significantly reduce the cost and technician labor of high through-put plasmid DNA purification for automated sequencing and mapping.
This work is being supported by DOE SBIR Phase II Grant # DE FG 03 - 94ER81802 /A000, W. MacConnell P.I.