Progress 10/01/01 to 09/30/07
Outputs
OUTPUTS: The reporting period includes a year of sabbatical work at the National Center for Biosafety in Norway. Prior approaches, which highlighted the specific removal of PCR inhibitors through protease treatment were discarded when we realized that this could not be utilized widely. Instead, a new approach was established, a non-specific, alkali-based pretreatment of individual particles from air samples which rendered both extraction and inhibition problems a moot point. Thus in Norway we succeeded in performing single-particle PCR amplifications which were enough to provide a clean and bright signal using standard and low-cost reagents. This method was further developed to the point where we now have a preliminary publication, through a patent, under discussion with the University of California's Office of Intellectual Property (IPIRA) and their Norwegian counterpart. Further elements of a possible device are now in draft and word description and will be submitted pending discussions on the patents. Meanwhile we continue to perform field sampling using standard air sampling methods across various transects. We consider this important to lay down baseline data for future mapping since the development of the method has taken such a long time. One graduate student successfully defended the concepts behind this effort in his preliminary exam, and another student was accepted in Berkeley to undertake the mapping and database mathematical aspects of the project. PARTICIPANTS: In addition to Ignacio Chapela, PI, the project included for this reporting period Dr. Terje Traavik, Director of the Center for Biosafety, Norway -who is also cited as co-inventor in a forthcoming patent application- and Ali B Tonak, graduate student at the Chapela lab and for a semester in Norway. TARGET AUDIENCES: An unexpected new "audience" can be identified in this reporting period, the audience of technology transfer and patent office specialists. This new group of readers comes in addition to the expected audience of farmers, city-managers, etc. as described before for this project. PROJECT MODIFICATIONS: We took a major new technical approach which allowed the non-specific, single-particle amplification of specific DNA sequences through PCR.
Impacts
A patent application is in discussion between the IPIRA office at the University of California and the Technology Transfer office in its Norwegian counterpart at the Norwegian Center for Biosafety. A PhD student successfully defended the concepts behind this effort in his preliminary exam, and another student was accepted in Berkeley to undertake the mapping and database mathematical aspects of the project.
Publications
- No publications reported this period
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Progress 01/01/05 to 12/31/05
Outputs
In this period, the research laboratory went through major reconfiguration and reorganizing. Research was continued to establish an in-situ PCR protocol to enable the use of sequence-specific visualization of DNA on individual particles (spores, pollen). No publications to report.
Impacts
The expected impact continues to be highly promising. In contrast to current methods of identification, which require each sample to be processed separately in test-tubes, the protocols devised under this project will allow each individual particle to operate, in practice, as an individual test. This will be useful not only for transgenic and biosafety research, but also for other fields.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
No progress to report for this period.
Impacts
This research should lead to a fast, comprehensive method for the direct detection of transgenic DNA in the environment. The level of detection, and the equipment being used should make it possible to process 2-4 orders of magnitude more individual genomes than it is possible to date. In addition, this method should allow for the detection of transgenic DNA by local individuals or communities, allowing for generalized, low cost, extensive monitoring urgently needed but not available today.
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
New primers were designed to allow for the fluorescence-detection of transgenic DNA in various microscopic particulates. A method was established to collect, fix and process particulates for visualization of fluroescent probe for detection. A first run was tested using the new COPAS particle fluorescence analyser. Conditions were established for the amplification of DNA within particles using polymerase chain reaction (PCR). A collection of particles from environmental samples was initiated to test age effect on the detection method. Relatively high background fluorescence was identified as a potential problem, although new tests using fluorochromes in a different spectral range promise to ameliorate this problem. In optimizing the direct amplification from particles, an in-vitro set of experiments identified a heat-stable, protease-sensitive, factor in pollen extracts that inhibits DNA polymerase activity in a dose-dependent fashion. We postulate that this factor
represents a protein in these particles that could potentially preclude the possibility of amplification using PCR. New fixation and pre-incubation methods are being tested to eliminate this inhibition ifor the field-based assays
Impacts
This research should lead to a fast, comprehensive method for the direct detection of transgenic DNA in the environment. The level of detection, and the equipment being used should make it possible to process 2-4 orders of magnitude more individual genomes than it is possible to date. In addition, this method should allow for the detection of transgenic DNA by local individuals or communities, allowing for generalized, low cost, extensive monitoring urgently needed but not available today.
Publications
- No publications reported this period
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