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Combining a Novel Comet Assay Procedure with Automated Imaging and Analysis to Enable High Throughput Genotoxicity Testingダウンロード
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February 04, 2016
Authors: Brad Larson, Peter Banks, BioTek Instruments, Inc. Winooski, VT USA; Clare Whittaker, Sandra Woodgate, Trevigen, Inc., Gaithersburg, MD USA
Human contact to genotoxins is inevitable, as agents detrimental to our DNA are present in our environment, and also a result of our own cellular metabolism where they can contribute to cell death, gene mutations, and even cancer, disease and aging. At the same time, therapeutic approaches such as chemotherapy and radiation treatment rely on initiation of tumor cell genotoxicity to produce positive therapeutic outcomes. Efficiently and precisely repairing damaged DNA depends on the cellular DNA repair capacity. Therefore, quantifying DNA damage and accurately measuring the rate and extent of repair is critical in epidemiology, toxicology, drug development and other applications. The comet assay is well established for quantifying DNA damage in mammalian cells and is compatible with detection of a wide array of DNA damaging agents. In fact, it can be used to monitor the cellular DNA repair capacity for several different DNA repair pathways. The assay’s principle is migration of fragmented DNA in an electrophoresis gel, where intact DNA (comet head) moves at a slower rate than fragmented DNA (comet tail) as evaluated via fluorescence staining and microscopy6. The percentage of fragmented DNA in the comet tail is a direct measure of DNA damage.
Comet assays are typically performed and analyzed manually, so although the assay provides increased sensitivity, adoption as a reliable method has been slow. Here we demonstrate a combined process to generate automated, high-throughput comet-based genotoxicity data. High-density slides allow 96 individual samples to be compared simultaneously. Following staining, automated imaging and analysis of each well is performed using a novel cell imaging multi-mode reader with commonly accepted calculations; such as percent DNA in the tail. The combination of a novel single-cell microarray comet assay format, and automated instrumentation and analysis, provides an accurate, robust method to assess DNA damage and DNA repair in mammalian cells.
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