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DETECTION OF DNA DAMAGE USING MELTING ANALYSIS TECHNIQUES
Citation:
Rogers, K R., K Ramanathan, AND A. B. Apostol. DETECTION OF DNA DAMAGE USING MELTING ANALYSIS TECHNIQUES. Presented at Chemical Sensors and Interfacial Design Conference, Ciocco, Italy, May 6-11, 2001.
Impact/Purpose:
The overall objective of this task is to develop scientifically sound sampling and bioanalytical approaches for screening and monitoring of hazardous wastes. These techniques are expected to provide the Agency with improved screening and field portable methods to characterize, reduce, and control risk to human health and the environment. Specific objectives will include development and characterization of the following concepts:
SPMDs for passive accumulation of TICs
Bioassays for toxic and genotoxic compounds
MIPs for volatile and semivolatile toxic organics
Rapid screening assays using the previously listed components.
Description:
A rapid and simple fluorescence screening assay for UV radiation-, chemical-, and enzyme-induced DNA damage is reported. This assay is based on a melting/annealing analysis technique and has been used with both calf thymus DNA and plasmid DNA (puc 19 plasmid from E. coli). DNA damage resulting from exposure to UV-C and UV-A radiation, several restriction endonucleases, and several classes of DNA damaging compounds can be measured. Based on a similar melting/annealing analysis concept, a rapid and sensitive fiber optic biosensor assay for ionizing radiation-induced DNA damage is also reported. For this 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 damage resulting from exposure of the dye-labeled strand to low doses of ionizing radiation. Ten assays (each requiring about 15 min) could be run on the same fiber. Radiation exposures of between 0.2 and 10 Gray (Gy) were measured.
The U.S. Environmental Protection Agency (EPA), through its Office of Research and Development (ORD), has funded this research and approved this abstract as a basis for a presentation.