You are here:
DETECTION OF LOW DOSE RADIATION-AND CHEMICALLY-INDUCED DNA DAMAGE USING TEMPERATURE DIFFERENTIAL FLUORESCENCE ASSAYS
Citation:
Rogers, K R., A. B. Apostol, O. S. Madsen, AND C. W. Spencer. DETECTION OF LOW DOSE RADIATION-AND CHEMICALLY-INDUCED DNA DAMAGE USING TEMPERATURE DIFFERENTIAL FLUORESCENCE ASSAYS. Presented at 8th International Meeting on Chemical Sensors, Basel, Switzerland, July 2-5, 2000.
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:
Rapid, sensitive and simple assays for radiation- and chemically-induced DNA damage can be of significant benefit to a number of fields including radiation biology, clinical research, and environmental monitoring. Although temperature-induced DNA strand separation has been used as an indicator of oxidative DNA damage and single strand breaks, recent advances in double strand-specific dyes and fiber optic biosensor techniques form the basis for improvements in these assays. Rapid and sensitive biosensor and optical screening assays that measure DNA damage resulting from chemical exposure or low dose radiation are reported. For the fiber optic biosensor assay, a biotin-labeled capture oligonucleotide (38 mer) was immobilized to an avidin-coated quartz fiber. Hybridization of a dye-labeled complementary sequence was observed using the evanescent wave and reported at the proximal end of the optical fiber. Changes in temperature-induced strand separation were then used as a sensitive indicator of DNA damage resulting from exposure of the dye-labeled strand to either SIN-1 (3-morpholinosydnonimine, a source of OH- radicals) or low doses of ionizing radiation. For the optical screening assay, changes in temperature induced strand separation in both calf thymus DNA and plasmid DNA (puc 19 plasmid from E. coli) were measured after exposure to low doses of ionizing radiation (0.004 to 1 Gray). The double strand sensitive dye PicoGreen was used as an indicator of DNA denaturation. Calibration plots indicate that fluorescence changes corresponding to amounts as low as 1 ng double stranded DNA (106 copies for plasmid puc19) are detected by this method.
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.