In Situ Detection of DNA Damage: Methods and Protocols (Methods in Molecular Biology, Vol 203) (Methods in Molecular Biology)

Detection and analysis of DNA damage is critically important in the study of the widest variety of biological phenomena, ranging from apoptosis and aging to heart diseases and anticancer drug development. In In Situ Detection of DNA Damage: Methods and Protocols, Vladimir Didenko and a panel of experts describe all the major in situ techniques for studying DNA damage and apoptosis, and show how approaches originally designed to label apoptotic cells can be used for DNA damage analysis (and vice versa). The readily reproducible techniques presented here include all those designed to detect specific single- and double-stranded DNA breaks in tissue sections while using polymerases, ligases, nucleases, and kinases. In-depth protocols for single-cell electrophoresis (comet assay) are presented, as well as those for the detection of indirect markers of DNA damage and modified bases. Technical reviews discuss specificity, sensitivity, advantages, and limitations of the techniques described, and compare them with alternative approaches. State-of-the-art and highly practical, In Situ Detection of DNA Damage: Methods and Protocols presents for the first time a complete set of enzymatic methods for determining DNA breaks, and will greatly facilitate the work of all those studying not only the in situ detection of DNA damage but also the labeling of apoptotic cells. Pt. 1. Labeling Dna Breaks Using Terminal Transferase (tunel Assay) -- Labeling Dna Damage With Terminal Transferase: Applicability, Specificity, And Limitations / P. Roy Walker -- Tunel Assay: An Overview Of Techniques / Deryk T. Loo -- Electron Microscopic Detection Of Dna Damage Labeled By Tunel / Antonio Migheli -- Quanitative Differentiation Of Both Free 3' Oh And 5' Oh Dna Ends Using Terminal Transferase-based Labeling Combined With Transmission Electron Microscopy / Yoshinori Otsuki -- Determination Of Three-dimensional Distribution Of Apoptotic Dna Damage By Combination Of Tunel And Quick-freezing And Deep-etching Techniques / Shinichi Ohno -- In Situ Detection Of Dna Strand Breaks In Analysis Of Apoptosis By Flow- And Laser-scanning Cytometry / Zbigniew Darzynkiewicz -- Pt. 2. Labeling Dna Breaks Using Dna Polymerase I Or Its Klenow Fragment --^ Dna Damage Detection Using Dna Polymersase I Or Its Klenow Fragment: Applicability, Specificity, Limitations / Jan Hein Van Dierendonck -- Labeling Dna Breaks In Situ By Klenow Enzyme / Katherine A. Wood -- In Situ Nick Translation At The Electron Microscopic Level / Marc Thiry -- Pt. 3. Labeling Dna Breaks Using Ligase -- In Situ Dna Ligation As A Method For Labeling Apoptotic Cells In Tissue Sections: An Overview / Peter J. Hornsby -- Detection Of Specific Double-strand Dna Breaks And Apoptosis In Situ Using T4 Dna Ligase / Vladimir V. Didenko -- In Situ Detection Of Double-strand Dna Breaks With Terminal 5'oh Groups / Vladimir V. Didenko -- Pt. 4. Detection Of Dna Breaks In Agarose Trapped Cells: Comet Assay And Related Techniques -- The Comet Assay: Principles, Applications, And Limitations / Andrew R. Collins -- The Comet Assay: An Overview Of Techniques / Peggy L. Olive --^ Ultrasensitive Detection Of Dna Damage By The Combination Of The Comet And Tunel Assays / Andrei L. Kindzelskii -- Application Of Fish To Detect Dna Damage: Dna Breakage Detection-fish (dbd-fish) / José Luis Fernández -- Pt. 5. Detection Of Modified Bases And Ap Sites In Dna -- Simultaneous In Situ Detection Of Dna Fragmentation And Rna/dna Oxidative Damage Using Tunel Assay And Immunohistochemical Labeling For 8-hydroxy-2'-deoxyguanosine (8-ohdg) / Alexander E. Kalyuzhny -- The In Situ Detection Of Apurinic/apyrimidinic Sites And Dna Breaks Bearing Extension Blocking Termini / Philip K. Liu -- Pt. 6. Indirect And General Markers Of Dna Damage -- Markers Of Poly (adp-ribose) Polymerase Activity As Correlates Of Dna Damage / Yinong Zhou -- Ultrasound Imaging Of Apoptosis: Dna-damage Effects Visualized / Gregory J. Czarnota -- P53 Induction As An Indicator Of Dna Damage / Galina Selivanova --^ -detection Of Caspases Activation In Situ By Fluorochrome-labeled Inhibitors Of Caspases (flica) / Zbigniew Darzynkiewicz. Edited By Vladimir V. Didenko. Includes Bibliographical References And Index.

In In Situ Detection of DNA Damage: Methods and Prools, Vladimir Didenko and a panel of experts describe all the major in situ techniques for studying DNA damage and apoptosis, and how approaches originally designed to label apoptotic cells can be used for DNA damage analysis (and vice versa). The readily reproducible techniques presented here include all those designed to detect specific single- and double-stranded DNA breaks in tissue sections while using polymerases, ligases, nucleases, and kinases. There are also novel instrumental techniques that use ultrasound for in vivo and in situ detection of apoptotic DNA damage, and in situ applications of flow and laser-scanning cytometry for analysis of DNA strand breaks and apoptosis. State-of-the-art and highly practical, In Situ Detection of DNA Damage: Methods and Prools presents for the first time a complete set of enzymatic methods for determining DNA breaks, and will greatly facilitate the work of all those studying the in situ detection of DNA damage and the labeling of apoptotic cells.

Front Matter....Pages i-xvii Front Matter....Pages 1-1 Front Matter....Pages 3-19 Front Matter....Pages 21-30 Front Matter....Pages 31-39 Front Matter....Pages 41-54 Front Matter....Pages 55-68 Front Matter....Pages 69-75 Back Matter....Pages 79-79 ....Pages 81-108 Apoptotic and programmed cell death are characterized by, and indeed were first discovered from observations of, remarkable morphological changes that occur in the nucleus (see 1 for a comprehensive review of apoptosis and programmed cell death).