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A FLUORESCENCE BASED ASSAY FOR DNA DAMAGE: INDUCED BY RADIATION, CHEMICALS AND ENZYMES
Citation:
Ramanathan, K, A. B. Apostol, K R. Rogers, AND R. Gary. A FLUORESCENCE BASED ASSAY FOR DNA DAMAGE: INDUCED BY RADIATION, CHEMICALS AND ENZYMES. Presented at American Chemical Society, Orlando, FL, April 7-11, 2002.
Impact/Purpose:
The overall objective of this task is to develop rapid, cost-effective and scientifically sound techniques for measuring chemically induced DNA damage. This method is expected to provide the Agency with rapid, sensitive, and simple techniques that can be used among a panel of methods to determine the genotoxic potential of polluted samples.
Description:
A simple and rapid assay to detect DNA damage is reported. This assay is based on the ability of certain dyes to fluoresce upon intercalation with dsDNA. Damage caused by ultraviolet (UV) radiation, chemicals or restriction enzymes is detected using this assay. UV radiation at 254 nm approximating UV-C and radiation at 360 nm approximating UV-A, were used to induce the damage in plasmid dsDNA (pUC19). Chemical damage was induced using several compound classes with known effects on nucleic acids. Restriction enzymes hind III, msp1, sau 3A1 were used to cut the plasmid at specific sequences in addition to the nonspecific endonuclease DNAase I. The effect of these types of damages on repeated melting and annealing of dsDNA were observed in real time using several fluorescence indicator dyes. The assay response for dsDNA between 10 and 100 ng/mL was linear with a detection limit of 20 pg and a coefficient of variation of 2% (CV%). The assay was also optimized to study the efficiency of various sun blocking agents against DNA damage.
The U.S. Environmental Protection Agency (EPA), through its Office of Research and Development (ORD), funded this research and approved this abstract as a basis for an oral presentation. The actual presentation has not been peer reviewed by EPA.