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A FLUORESCENCE BASED ASSAY FOR DNA DAMAGE INDUCED BY RADIATION, CHEMICAL MUTAGENS AND ENZYMES
Citation:
Ramanathan, K, R. Gary, A. B. Apostol, AND K R. Rogers. A FLUORESCENCE BASED ASSAY FOR DNA DAMAGE INDUCED BY RADIATION, CHEMICAL MUTAGENS AND ENZYMES. CURRENT APPLIED PHYSICS 3(2-3):99-106, (2003).
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 novel assay is based on changes in melting/annealing behavior and facilitated using certain dyes that increase their fluorescence upon association with double stranded (ds)DNA. Damage caused by ultraviolet (UV) radiation, chemical mutagens or restriction enzymes produced an assay response. UV radiation at 254 nm (approximating UV-C) and 360 nm (approximating UV-A) were used to induce the damage in dsDNA. Chemical damage was induced using several compounds with known effects on nucleic acids. Restriction enzymes Hind III, Msp1, Sau 3A1 were used to cut the plasmid (pUC19) at specific sequences in addition to the nonspecific endonuclease DNase I. The effects of these types of damage on repeated melting and annealing of dsDNA were observed in real time using several fluorescence indicator dyes. Low concentrations of dsDNA (between 10 and 100 ng/mL) and small volumes (20 uL) were required for this assay. Repeated measures yielded a coefficient of variation of 2% (CV%). In addition to measuring various DNA damaging agents, the potential application of this assay to study the efficiency of various sun blocking agents against UV-induced DNA damage is discussed.
The U.S. Environmental Protection Agency (EPA), through its Office of Research and Development (ORD), funded this research. It has been subjected to the EPA's peer and administrative review and approved for publication.