DOE Human Genome Program Contractor-Grantee
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
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
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.
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