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This volume describes all the major in situ techniques for studying DNA damage and apoptosis, and demonstrates how approaches originally designed to label apoptotic cells can be applied to DNA damage analysis (and vice versa). All the techniques using polymerases, ligases, nucleases, and kinases to detect specific single- and double-stranded DNA breaks are described. Technical reviews discuss specificity, sensitivity, advantages, and limitations of the techniques described, and compare them with alternative approaches.Book News, Inc

Book Info

Baylor College of Medicine, Houston, TX. Includes all major techniques in the field, new in situ applications of flow and laser-scanning cytometry, detailed in situ ligation protocols, and describes the use of ultrasound for in vivo and in situ detection of DNA damage. DNLM: DNA Damage.

Book Description

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 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 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 the in situ detection of DNA damage and the labeling of apoptotic cells.

Preface v
Contributors xiii
Color Plates xvii
Part I Labeling DNA Breaks Using Terminal Transferase (TUNEL Assay)
1 Labeling DNA Damage with Terminal Transferase: Applicability, Specificity, and Limitations 3
2 TUNEL Assay: An Overview of Techniques 21
3 Electron Microscopic Detection of DNA Damage Labeled by TUNEL 31
4 Quantitative Differentiation of Both Free 3' OH and 5' OH DNA Ends Using Terminal Transferase-Based Labeling Combined with Transmission Electron Microscopy 41
5 Determination of Three-Dimensional Distribution of Apoptotic DNA Damage by Combination of TUNEL and Quick-Freezing and Deep-Etching Techniques 55
6 In Situ Detection of DNA Strand Breaks in Analysis of Apoptosis by Flow- and Laser-Scanning Cytometry 69
Part II Labeling DNA Breaks Using DNA Polymerase I or its Klenow Fragment
7 DNA Damage Detection Using DNA Polymerase I or its Klenow Fragment: Applicability, Specificity, Limitations 81
8 Labeling DNA Breaks In Situ by Klenow Enzyme 109
9 In Situ Nick Translation at the Electron Microscopic Level 121
Part III Labeling DNA Breaks Using Ligase
10 In Situ DNA Ligation as a Method for Labeling Apoptotic Cells in Tissue Sections: An Overview 133
11 Detection of Specific Double-Strand DNA Breaks and Apoptosis In Situ Using T4 DNA Ligase 143
12 In Situ Detection of Double-Strand DNA Breaks with Terminal 5'OH Groups 153
Part IV Detection of DNA Breaks in Agarose Trapped Cells: Comet Assay and Related Techniques
13 The Comet Assay: Principles, Applications, and Limitations 163
14 The Comet Assay: An Overview of Techniques 179
15 Ultrasensitive Detection of DNA Damage by the Combination of the Comet and TUNEL Assays 195
16 Application of FISH to Detect DNA Damage: DNA Breakage Detection-FISH (DBD-FISH) 203
Part V Detection of Modified Bases and AP Sites in DNA
17 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) 219
18 The In Situ Detection of Apurinic/Apyrimidinic Sites and DNA Breaks Bearing Extension Blocking Termini 235
Part VI Indirect and General Markers of DNA Damage
19 Markers of Poly (ADP-Ribose) Polymerase Activity as Correlates of DNA Damage 247
20 Ultrasound Imaging of Apoptosis: DNA-Damage Effects Visualized 257
21 p53 Induction as an Indicator of DNA Damage 279
22 Detection of Caspases Activation In Situ by Fluorochrome-Labeled Inhibitors of Caspases (FLICA) 289
Index 301