Development of a standard high throughput comet assay

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DESCRIPTION (provided by applicant): The single cell gel electrophoresis (SCGE) or comet assay is now recognized worldwide as a simple and inexpensive method for detection of different types of DNA damage. Adoption of the comet assay for genotoxic studies has been hampered to date by the lack of a standardized platform and procedures for the assay, as well as the labor-intensive nature of the assay for multiple-sample analysis. Another problem associated with the assay is that to avoid introduction of artificial DNA damage to cells, the assay needs to be run of freshly isolated cells or tissues. This make it difficult to direct compare cells collected at various times, or to collect samples in the field where the assay cannot immediately performed. Trevigen in the Phase I application developed a high throughput comet slides for standardization of the comet slide preparation for the assay. These slides are available at present in formats for single assay analysis (2 well format) and also for high throughput analysis (20 and 96 well format). Trevigen also demonstrated in preliminary studies that it is possible to preserve cells without DNA damage for analysis later or to use as standards in subsequent assays. The goals of the Phase II application are: 1. To standardize the electrophoretic platform and procedures for the comet assay by developing a controlled environment for electrophoresis of the damaged DNA in the cells, which will eliminate the problems, associated with reproducibility of the comet assay in the day to day operation. 2. To develop comet assay standards based on cells, which were treated with various concentration of DNA damaging agents, and long term preservation of these standards or other biological materials, to be used as reference material in the comet assay. The accomplishment of these aims will results in a standardized comet assay procedure, which can be used in any laboratory and can be developed as a high throughput automatic system. The preservation of cells without DNA damage will allow development of standards for the comet assay and will allow that biological material could be archived for future studies or to be shared between laboratories. It would also allow field collected samples to be safely transported for analysis later. The single cell gel electrophoresis (SCGE) or comet assay is a simple and inexpensive method for detection of different types of DNA damage. The long-term objective of this project is to develop standardized platform and procedures, including DNA standards for this assay.