DOE Human Genome Program Contractor-Grantee Workshop VIII
February 27-March 2, 2000  Santa Fe, NM

42. Automated, Low Cost Isolation of Blood or Bacterial Genomic DNA

Brian Bauman, Tuyen Nguyen, Zuxu Yao, Tony Zucca, Dan Langhoff, and William MacConnell

MacConnell Research Corporation, San Diego, CA 92121

macres@macconnell.com

The isolation of the genomic DNA from blood, bacteria, and virus is a necessary starting point for molecular diagnosis of infection, genetic disease, inherited traits and identity determination, and other research applications. The ability to rapidly and reproducibly isolate DNA from blood and other bodily samples will be continually required to identify, characterize and treat factors involved in human disease and disorders. This process needs to be automated by means that are affordable to clinical or research labs. In Phase II we are further developing prototypes for a fully automatic high-throughput blood or bacteria genomic DNA isolation instrument and disposable processing cassettes. This instrument uses a derivative of electrophoretic separation technology that we developed for purification of plasmid DNA. Proof-of-concept data has been obtained during Phase I. The instrument gives high yields of pure DNA over a wide range of sample concentrations.

The separation technology makes use of programmed electrophoresis of the lysed sample which is placed in between boundaries of agarose separation material. The process can be accomplished using a microprocessor-controlled programmable power supply in conjunction with a multi-sample disposable processing cassette, electrophoresis rig and fluid handling components.

Thus far, our prototype instrument and cassettes allows automatic purification of human blood or bacterial DNA in as little as 24 minutes. The resulting DNA is pure enough for use in restriction digests, and could be used as a template for PCR, PCR sequencing and RFLP analysis. In addition, we found that the process successfully purifies genomic DNA from a wide range of sample cell numbers, such as 104 to 109 bacterial cells or 0.2 to 500 microliters of human blood. Interestingly, the DNA yield from the above trials was greater than 75% of the theoretical amount of genomic DNA present in these samples.

In Phase II we are completing trials to: (1) optimize the electrophoretic purification technique, (2) determine the full range of sample volumes and cell numbers that can be processed, (3) test the activity of purified genomic DNA in a variety of molecular biology procedures, (4) test for cross-contamination between samples purified in the same run, (5) construct a 24 well and 96 well prototype instruments that automate the overall method, (6) write control software and (7) contract for the construction of an injection mold for the production of the processing cassettes. The method is being expanded to process DNA from a variety of sample types including viruses.

The products developed in this work will be commercialized by MacConnell Research in the form of instruments and supplies sold for genomic DNA isolation.

This research is being supported by DOE SBIR Phase II grant number DE-FG03-98ER82612.