In Situ Labeling of DNA Breaks and Apoptosis by T7 DNA Polymerase

Didenko, Vladimir V.

doi:10.1007/978-1-60327-409-8_4

Cited by 6 publications

(2 citation statements)

References 31 publications

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“…Recently, a further modification of this assay, Repair Assisted Damage Detection (RADD) was developed with a refined enzyme cocktail for the detection of abasic sites, uracils, strand breaks, and oxidative and UV-induced DNA lesions within cells (Fig. 2) These DNA repair enzyme-mediated damage detection strategies offer significant improvements over other enzyme detection strategies such as terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) [35][36][37]. TUNEL has been employed to detect DNA strand breaks, typically in the context of apoptosis, but also in some cases DNA adducts across a variety of biological samples [38][39][40].…”

Section: Enzyme-mediated Damage Detection Assaysmentioning

confidence: 99%

Targets for repair: detecting and quantifying DNA damage with fluorescence-based methodologies

Gassman

Holton

2019

Current Opinion in Biotechnology

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Detection and characterization of DNA damage is essential for evaluating genotoxicity, monitoring DNA repair, developing biomarkers for exposures, and evaluating the efficacy of chemotherapies. These diverse applications for DNA damage measurements have spurred the continual development and refinement of methodologies for detecting, characterizing, and quantifying DNA damage from isolated DNA and in cells and tissues. Current damage detection methods cover a wide range of techniques from radiolabeling to mass spectrometry, and use of these techniques varies widely based on expense, expertise, and knowledge of adduct formation. More generalizable, easy-to-use methods for detecting and quantifying DNA damage are needed, and there has been an emergence of fluorescence-based methodologies to address this need. Developments in these fluorescence-based strategies are reviewed here.

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Section: Enzyme-mediated Damage Detection Assaysmentioning

confidence: 99%

Targets for repair: detecting and quantifying DNA damage with fluorescence-based methodologies

Gassman

Holton

2019

Current Opinion in Biotechnology

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“…Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and in situ (DNA) end labeling (ISEL) [16–18] have also been employed extensively over the past 20 years to detect DNA strand breaks during apoptosis and in some cases DNA damage across a variety of biological samples [19–21]. However, just like the other methods, there are drawbacks to using TUNEL or ISEL because they are highly specific for 3’-OH ends.…”

Section: Introductionmentioning

confidence: 99%

Broad spectrum detection of DNA damage by Repair Assisted Damage Detection (RADD)

2018

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Environmental exposures, reactive by-products of cellular metabolism, and spontaneous deamination events result in a spectrum of DNA adducts that if un-repaired threaten genomic integrity by inducing mutations, increasing instability, and contributing to the initiation and progression of cancer. Assessment of DNA adducts in cells and tissues is critical for genotoxic and carcinogenic evaluation of chemical exposure and may provide insight into the etiology of cancer. Numerous methods to characterize the formation of DNA adducts and their retention for risk assessment have been developed. However, there are still significant drawbacks to the implementation and wide-spread use of these methods, because they often require a substantial amount of biological sample, highly specialized expertise and equipment, and depending on technique, may be limited to the detection and quantification of only a handful of DNA adducts at a time. There is a pressing need for high throughput, easy to implement assays that can assess a broad spectrum of DNA lesions, allowing for faster evaluation of chemical exposures and assessment of the retention of adducts in biological samples. Here, we describe a new methodology, Repair Assisted Damage Detection (RADD), which utilizes a DNA damage processing repair enzyme cocktail to detect and modify sites of DNA damage for a subsequent gap filling reaction that labels the DNA damage sites. This ability to detect and label a broad spectrum of DNA lesions within cells, offers a novel and easy to use tool for assessing levels of DNA damage in cells that have been exposed to environmental agents or have natural variations in DNA repair capacity.

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Methods to detect apoptotic cell death

Bánfalvi

2016

Apoptosis

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In concert with the increased understanding that there are many ways for cells to die, several methods have been developed to detect cell death. The classification of cell death posed some difficulties that were overcome by implementing strict selection criteria that should also apply to the detection methods. The selection of assays is based on morphological criteria and distinguishable marks of apoptotic patways. The detection of apoptosis includes methods related to membrane alterations, DNA fragmentation, cytotoxicity and cell proliferation, mitochondrial damage, immunological detection and mechanism based assays. Other less frequently used detections of apoptosis are: (a) light-scattering flow cytometry to avoid underestimating the extent and timing of apoptosis, (b) time-lapse microscopy perfusion platform to support the temporal aspects of detection, to measure cell surface area and cellular adhesion, and (c) genotoxicity specific chromatin changes. Attention is called to the advantages and limitations of various methods.

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In Situ Labeling of DNA Breaks and Apoptosis by T7 DNA Polymerase

Cited by 6 publications

References 31 publications

Targets for repair: detecting and quantifying DNA damage with fluorescence-based methodologies

Targets for repair: detecting and quantifying DNA damage with fluorescence-based methodologies

Broad spectrum detection of DNA damage by Repair Assisted Damage Detection (RADD)

Methods to detect apoptotic cell death

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