Titlebook: In Situ Detection of DNA Damage; Methods and Protocol Vladimir V. Didenko Book 20021st edition Springer Science+Business Media New York 200

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Determination of Three-Dimensional Distribution of Apoptotic DNA Damage by Combination of TUNEL and lly defined by its unique ultrastructural features, which were detected by electron microscopy (.,.). They are cytoplasmic shrinkage, nuclear chromatin condensation along nuclear margin, cell fragmentation into apoptotic bodies, and phagocytosis by adjacent epithelial cells or macrophages. It is als

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Detection of DNA Strand Breaks in Analysis of Apoptosis by Flow- and Laser-Scanning Cytometryristic features of apoptotic cells (.,.). A widely used methodology to detect apoptotic cells, thus, relies on labeling DNA strand breaks in situ, within the nuclear chromatin, either with fluorochromes (.–.) or absorption dyes (.–.). The overview of the techniques (TUNEL techniques), which were dev

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DNA Damage Detection Using DNA Polymerase I or its Klenow Fragmentf cell death, during the past few years polymerase-based assays for detection of DNA breaks in cellular nuclei have found worldwide application in many fields of cellular biology and medicine. This interest has greatly stimulated research on technical improvements of the assays and has led to the av

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Labeling DNA Breaks , by Klenow Enzymeerg polymerase of .. Klenow is now usually obtained as a recombinant protein expressed from a truncated . gene (.). To polymerize the addition of nucleotides to the 3′-OH end of DNA, Klenow requires a single stranded DNA template and a DNA or RNA primer with a 3′−hydroxyl terminus. Klenow lacks the

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Nick Translation at the Electron Microscopic Levelies on combined 5′→3′ polymerase and 5′→3′ exonuclease activities of . DNA polymerase I. If a nick or single strand break with a 3′ hydroxyl terminus is present in a duplex DNA molecule, the enzyme will translocate it, removing nucleotides ahead of it by 5′→3′ exonuclease activity and synthesizing t

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DNA Ligation as a Method for Labeling Apoptotic Cells in Tissue Sectionshe ability of terminal deoxyribonucleotidyl transferase to add nucleotides to breaks in DNA, and has generally been termed TUNEL (terminal dUTP nick end labeling), although ISEL (. end labeling) would be a more appropriate description. During the late stages of apoptosis, nucleases are activated tha

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Detection of Specific Double-Strand DNA Breaks and Apoptosis , Using T4 DNA LigaseYet historically, the labeling of DNA breaks . was most often limited to the identification of apoptotic cells. Consequently, the major techniques for analysis of DNA cleavage in tissue sections were initially developed for the visualization of apoptotic death. These assays rely on enzymatic labelin

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The Comet Assay applications in genototoxicity testing, human biomonitoring and molecular epidemiology, ecotoxicology, as well as fundamental research in DNA damage and repair. The assay attracts adherents by its simplicity, sensitivity, versatility, speed and economy, and the number of publications it spawns rise

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The Comet Assayimplest form, cells are embedded in agarose on a microscope slide, immersed in a lysis solution to remove lipids and proteins, and exposed to a weak electric field to attract broken, negatively-charged DNA towards the anode. After electrophoresis, DNA is stained using a fluorescent dye, and viewed u