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

Detection and analysis of DNA damage is of critical importance in a variety of biological disciplines studying apoptosis, cell cycle and cell di- sion, carcinogenesis, tumor growth, embryogenesis and aging, neu- degenerative and heart diseases, anticancer drug development, environmental and radiobiological research, and others. Individual cells within the same tissue or in cell culture may vary in the extent of their DNA damage and, consequently, can display different re- tions to it. These differences between individual cells in the same cell popu- tion are detected using in situ approaches. In situ is a Latin term meaning “on site” or “in place.” It is used to denote the processes occurring or detected in their place of origin. In mole- lar and cell biology this usually refers to undisrupted mounted cells or tissue sections. In that meaning “in situ” is used as part of the terms “in situ PCR,” “in situ transcription,” “in situ hybridization,” “in situ end labeling,” and “in situ ligation.” Sometimes the “in situ” term is applied at the subcellular level to cells disrupted in the process of analysis, for example, in the detection of specific sequences in chromosomes using fluorescent in situ hybridization (FISH). Historically, the term was used primarily in methods dealing with nucleic acids.

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.