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Polymerase chain reaction (PCR) is an in vitro enzymatic, synthetic method used to amplify specific DNA sequences from organisms. Detection of target DNA using gene probes allows for absolute identification not only of specific organisms, but also of genetic material in recombinant organisms. A major challenge facing detection of DNA from field samples is that they are almost sure to contain impurities, especially impurities that inhibit amplification through PCR. For example, humic material even in quantities as small as 1 ng have been shown to inhibit PCR. DNA has been extracted from sewage/stool samples, food, sputum, water and sediment, and human DNA likewise is being extracted from sources as varied as forensic samples of blood, cigarette butts and human remains. However, multi-step, time consuming methods are required to isolate the DNA from the surrounding contamination. This research focuses on developing a method for rapid cleanup of DNA which combines extraction and purification of DNA while, at the same time, removing inhibitors from ‘dirty samples’ to produce purified, PCR-ready DNA. GeneReleaser™ produces PCR-ready DNA in a rapid 5-min protocol. We report here the rapid extraction/purification of plasmid DNA from recombinant Escherichia coli . GeneReleaser™, inhibitor and ∼10 3 cfu recombinant E. coli containing a plasmid insert were added to PCR tubes, vortexed for 30 s and microwaved for 5 min. DNA was PCR-amplified and identified by gel electrophoresis. GeneReleaser™ (GR) resin was able to cleanup samples containing typical aerosol and water/soil contaminants (dust—60 μg, pollen—100 μg, soot—250 μg, humic acid—75 ng). While these inhibitors were easily removed prior to PCR amplification, other complex inhibitors found in soil and food samples remain a major challenge and detection of DNA in these materials typically requires multi-step procedures that may take up to a day. The advantages of using GR are that it is rapid and inexpensive.

A large semi-synthetic recombinant antibody library was constructed based on a human anti-tetanus library. The library was used to isolate a human anti-cholera toxin B antibody cidne, which is expressed in XL-1 Blue E. Coli bacteria. The clone, B-27 cholera Fab, was expressed in small scale and purified by a two column procedure, which uses the histidine affinity tag, which was engineered into the protein. The purified cholera Fab was tested by ELISA to confirm specificity and by surface plasmon resonance to determine affinity binding constants.

An antibody-based fiber optic biosensor is described. This biosensor will serve as a model for future development of antibody-based detection of biological toxins. It is sensitive, specific, and new assays will be based solely on the antibody recognition element of the sensor.

Biodegradation of electronic components was investigated from historical and experimental standpoints. A literature review was conducted on new materials. Twisted wire pairs were tested for degradation by decontamination solution (DS-2). Capacitance readings were used to determine degradation. Initial readings of the immersed wires dropped to zero, indicating shorting of the wire, followed by reading oscillation and return to approximately the initial reading. This phenomenon may be due to pinhole sealing. Further testing is necessary but will require the fabrication of a bridge capable of meeting ASTM/MIL STD specifications. (Author)