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A Fluorescence Based Assay for Dna Damage Induced By Radiation, Chemical Mutagens and Enzymes

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Abstract: 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.
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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).
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Contact: Liz Hope - (919) 541-2785 or hope.elizabeth@epa.gov
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Division: Human Exposure & Atmospheric Sciences Division
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Branch: Exposure & Dose Research Branch
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Product Type: Journal
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Published: 04/01/2003
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Bullet Item A Fluorescence Based Assay for Dna Damage Induced By Radiation, Chemical Mutagens and Enzymes
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Related Entries:
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Bullet Item Development of Rapid Techniques for Detection of Chemically-Induced Dna Damage
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