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> Physiology > Molecular Function > DNA Breaks

DNA Breaks

DNA breaks, also known as DNA double-strand breaks, are disruptions in the continuity of the DNA molecule.
These breaks can occur due to various factors, such as ionizing radiation, chemical agents, or cellular processes like DNA replication and repair.
Unrepaired or misrepaired DNA breaks can lead to genomic instability, cell death, or the development of genetic diseases, including cancer.
Understanding the mechanisms and effects of DNA breaks is crucial for advancing research in fields like molecular biology, genetics, and cancer biology.
Effectively identifying and evaluating DNA break research protocols can accelerate scientific discovery and inform the development of new therapeutic strategies.

Most cited protocols related to «DNA Breaks»

1
TUNEL Assay for Detecting DNA Strand Breaks
DNA strand breaks were demonstrated by labeling free 3′-OH termini with FITC-labeled deoxyuridine, which was detected with alkaline phosphatase–coupled, anti-fluorescein antibody, and the formation of a dye precipitate with a phosphatase substrate (In Situ Cell Death Detection Kit, AP; Boehringer Mannheim, Mannheim, Germany). Yeast cells were fixed with 3.7% formaldehyde, digested with lyticase, and applied to a polylysine-coated slide as described for immunofluorescence (Adams and Pringle, 1984 (link)). The slides were rinsed with PBS, incubated in permeabilization solution (0.1% Triton X-100, 0.1% sodium citrate) for 2 min on ice, rinsed twice with PBS, incubated with 10 μl TUNEL reaction mixture (200 U/ml terminal deoxynucleotidyl transferase, 10 mM FITC-labeled dUTP, 25 mM Tris/HCl, 200 mM sodium cacodylate, 5 mM cobalt chloride; Boehringer Mannheim) for 60 min at 37°C, rinsed three times with PBS, incubated with 50 μl Converter AP solution (alkaline phosphatase– labeled, anti-FITC antibody; Boehringer Mannheim) for 30 min at 37°C, rinsed three times with PBS, and stained by incubation with 50 μl naphthol) AS-MX phosphate (Sigma Chemical Co., Munich, Germany), 0.8 mg/ml, fast red TR salt (Sigma Chemical Co.), 1 mg/ml, 2% dimethylformamide, 1 mM levamisole in 100 mM Tris/HCl, pH 8.2, for 30 min at room temperature. A coverslip was mounted with a drop of Kaiser's glycerol gelatin (Merck, Darmstadt, Germany).
Madeo F., Fröhlich E, & Fröhlich K.U. (1997). A Yeast Mutant Showing Diagnostic Markers of Early and Late Apoptosis. The Journal of Cell Biology, 139(3), 729-734.
Publication 1997
Alkaline Phosphatase Antibodies, Anti-Idiotypic Cacodylate Cell Death Cells cobaltous chloride Deoxyuridine deoxyuridine triphosphate Dimethylformamide DNA Breaks DNA Nucleotidylexotransferase fast red TR salt Fluorescein Fluorescein-5-isothiocyanate Fluorescent Antibody Technique Formaldehyde Gelatins Glycerin In Situ Nick-End Labeling Levamisole Hydrochloride lyticase Naphthols Phosphates Phosphoric Monoester Hydrolases Polylysine Sodium Sodium Citrate Triton X-100 Tromethamine Yeast, Dried
2
Mouse Brain Irradiation Feasibility Studies

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Publication 2008
alexa fluor 488 Animals Antibodies, Anti-Idiotypic Brain DNA Breaks Goat Immunoglobulins Mice, House Microscopy, Fluorescence Molecular Probes Obstetric Delivery Paraffin Embedding paraform Phosphorylation POU3F2 protein, human Radiation Radiotherapy Vision X-Ray Computed Tomography
3
Cisplatin and MK-1775 Combination Therapy
HNSCC cells (50,000) were seeded in 60-mm dishes, treated the next day with 1.5 μmol/L cisplatin, 0.25 μmol/L MK-1775 either alone or in combination for 48 hours, and then harvested 0, 24, or 48 hours later. Cells were fixed in ice-cold 70% ethanol, permeabilized with 0.25% Triton X-100 in PBS, and incubated with phospho-Histone H3 (Ser10) antibody conjugated to Alexa Flour 488 (#9708; at concentration 1:100, from Cell Signaling Technology) for 2 hours at 4°C. DNA was stained with 20 μg/mL propidium iodide (PI; Sigma-Aldrich) in the presence of 100 μg/mL RNase A (Sigma-Aldrich). For apoptosis assessment, cells were treated as indicated above and DNA strand breaks detected with an APO-BrdU TUNEL assay kit (Life Technologies) according to the manufacturer’s instructions. Samples were analyzed on a Gallios Flow Cytometer (Beckman Coulter, Inc.) combined with Flo-Jo software (FloJo).
Osman A.A., Monroe M.M., Ortega Alves M.V., Patel A.A., Katsonis P., Fitzgerald A.L., Neskey D.M., Frederick M.J., Woo S.H., Caulin C., Hsu T.K., McDonald T.O., Kimmel M., Meyn R.E., Lichtarge O, & Myers J.N. (2014). Wee-1 Kinase Inhibition Overcomes Cisplatin Resistance Associated with High-Risk TP53 Mutations in Head and Neck Cancer through Mitotic Arrest Followed by Senescence. Molecular cancer therapeutics, 14(2), 608-619.
Publication 2014
Apoptosis Biological Assay Bromodeoxyuridine Cells Cisplatin Common Cold DNA Breaks Endoribonucleases Ethanol Flour Histone H3 Hyperostosis, Diffuse Idiopathic Skeletal Immunoglobulins In Situ Nick-End Labeling MK-1775 Propidium Iodide Squamous Cell Carcinoma of the Head and Neck Triton X-100
4
Automated FADU Assay for DNA Damage Analysis
This new version of the FADU assay comprises a total of 11 steps described below. It should be noted that steps 2 and 3 are alternatives, depending on whether or not DNA repair is to be analysed. Steps 1, 2, 3, 10 and 11 were performed manually whereas the critical steps 4–9 have been automated (Fig. 1A). It should be noted that in some experiments performed for protocol optimisation (Fig. 2A), different conditions were used, as indicated.
T samples represent the total amount of DNA present.B samples represent the background fluorescence observed when DNA is denatured completely. P0 samples reflect DNA strand interruptions present under physiological conditions. Px samples (P1, P2, P3 etc) are from cells to which DNA damage had been inflicted in order to induce DNA strand breaks, and Rx samples (R1, R2, R3 etc) are from cells allowed to repair DNA damage.
Moreno-Villanueva M., Pfeiffer R., Sindlinger T., Leake A., Müller M., Kirkwood T.B, & Bürkle A. (2009). A modified and automated version of the 'Fluorimetric Detection of Alkaline DNA Unwinding' method to quantify formation and repair of DNA strand breaks. BMC Biotechnology, 9, 39.
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Publication 2009
Biological Assay Cells DNA Breaks DNA Damage DNA Repair Fluorescence physiology
5
Ligation-mediated PCR for DNA break detection
Supernatant was removed from agarose plugs, replaced with 50 μl fresh 1× ligase buffer, and plugs were heated to 62°C to melt agarose. 20 μl DNA (∼10,000 cell equivalents) was added to 2 μl T4 DNA ligase (2 Weiss units, MBI Fermentis), 10 μl ds annealed linker in 1× ligase buffer, 3 μl 10× ligase buffer, and 30 μl dH20, and incubated overnight at 18°C. Linker was prepared by annealing 5 nmol each of LMPCR.1 (5′-GCGGTGACCCGGGAGATCTGAATTC-3′) and LMPCR.2 (5′-GAATTCAGATC-3′) in 300 μl 1× ligase buffer, which results in a ds oligo with a 14-nt ss overhang that can only ligate unidirectionally. Ligated DNA samples were heated to 70°C for 10 min, diluted fivefold in distilled H20, and assayed for GAPDH by PCR to adjust DNA input before LM-PCR. The following primers (Integrated DNA Technologies) were used in conjunction with linker primer (LMPCR.1) to amplify DNA breaks: 5′Sμ-GCAGAAAATTTAGATAAAATGGATACCTCAGTGG-3′ (used for all experiments except for data shown in Fig. S1); 3′Sμ: 5′-GCTCATCCCGAACCATCTCAACCAGG-3′ (used only for Fig. S1); Sg3-AP: 5′-AACATTTCCAGGGACCCCGGAGGAG-3′ (25 (link)); and CmuL2: 5′-CTGCGAGAGCCCCCTGTCTGATAAG-3′ (42 (link)).
Three-fold dilutions of input DNA (0.5, 1.5, and 4.5 μL) were amplified by Hotstar Taq (QIAGEN) using a touchdown PCR program. PCR products were run on 1.25% agarose gels and vacuum blotted (VacuGene XL, Pharmacia) onto nylon membranes (GeneScreen Plus, PerkinElmer). Blots were hybridized with oligonucleotide probes end-labeled with γ32P-ATP at 37°C overnight and washed at 55°C with 2X SSC/0.1%SDS. The following probes were used: μ probe 5′: 5′-AGGGACCCAGGCTAAGAAGGCAAT-3′; Sμ probe: 5′-GTTGAGAGCCCTAGTAAGCGAGGCTCTAAAAAGCACGCT-3′ (7 (link)); Sμ3′ probe: 5′-GGGCTGGCTGATGGGATGCCCC-3′ (used only for Fig. S1); Sγ3-LP: 5′-GGACCCCGGAGGAGTTTCCATGATCCTGGG-3′ (25 (link)); and Cμ: 5′-TGGCCATGGGCTGCCTAGCCCGGGACTTCCTG-3′ (42 (link)).
Schrader C.E., Linehan E.K., Mochegova S.N., Woodland R.T, & Stavnezer J. (2005). Inducible DNA breaks in Ig S regions are dependent on AID and UNG. The Journal of Experimental Medicine, 202(4), 561-568.
Publication 2005
Buffers Cells DNA Breaks GAPDH protein, human Gels Ligase Nylons Oligonucleotide Primers Oligonucleotide Probes Oligonucleotides Sepharose T4 DNA Ligase Technique, Dilution Tissue, Membrane Vacuum

Most recents protocols related to «DNA Breaks»

1
Quantification of Apoptotic Retinal Cells via TUNEL Assay
In situ cell death detection kit (Roche) based on labeling of DNA strand breaks [(TdT-mediated dUTP-X nick end labeling (TUNEL)] was used to quantify apoptotic retinal cells (Table 1, Additional file 1: Table S1). Briefly, the DNA cleavage can be detected by labeling the free 3′-OH termini with fluorescein modified nucleotides in an enzymatic reaction. According to the manufacturer’s protocol, retinal cell death detection was conducted using 14 µm retinal cryosections from 4% PFA (w/v) fixed eyes (methods described before). After three washes with PB, slides were incubated in phosphate buffered saline (PBS) with 1% Triton X-100 (v/v) for 5–10 min at RT in humidity chamber. Then, TUNEL mix reagent was incubated for 1 h at 37 °C in dark conditions. Thereafter, the reaction was stopped with three PB washes for 5 min at RT in the dark. Finally, preparations were mounted using Citifluor and coverslipped.
Albertos-Arranz H., Martínez-Gil N., Sánchez-Sáez X., Noailles A., Monferrer Adsuara C., Remolí Sargues L., Pérez-Santonja J.J., Lax P., Calvo Andrés R, & Cuenca N. (2023). Microglia activation and neuronal alterations in retinas from COVID-19 patients: correlation with clinical parameters. Eye and Vision, 10, 12.
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Publication 2023
Apoptosis Cell Death Cells Cryoultramicrotomy deoxyuridine triphosphate DNA Breaks DNA Cleavage Enzymes Eye Fluorescein Humidity In Situ Nick-End Labeling Nucleotides Phosphates Retina Saline Solution Triton X-100
2
Detecting Apoptotic Sensory Neurons
To identify DNA breaks, characteristic of apoptotic sensory neurons, paraffin-embedded ganglia from latently infected mice were deparaffinized and treated with citric acid for permeabilization and antigen retrieval. Sections were subjected to TUNEL assay (Roche; number 11684795910) according to the manufacturer’s recommendations and were costained with anti-N200 (neurofilament 200) antibody. Ganglia sections treated with 1,000 U/mL DNase I at 37°C for 15 min prior to the TUNEL reaction served as positive controls.
Arbuckle J.H., Vogel J.L., Efstathiou S, & Kristie T.M. (2023). Deletion of the Transcriptional Coactivator HCF-1 In Vivo Impairs the Removal of Repressive Heterochromatin from Latent HSV Genomes and Suppresses the Initiation of Viral Reactivation. mBio, 14(1), e03542-22.
Publication 2023
Antigens Apoptosis Biological Assay Citric Acid Deoxyribonuclease I DNA Breaks Ganglia Immunoglobulins In Situ Nick-End Labeling Mice, House neurofilament protein H Neuron, Afferent Paraffin Embedding
3
Comet Assay for Tyrosine Kinase Inhibitor Genotoxicity
The induction of DNA strand breaks after TKI exposure was assessed by the comet assay according to Møller et al. [23 (link)], with minor modifications [20 (link)]. Briefly, ZFL cells were seeded at a density of 100,000 cells/well onto 12-well cell culture plates (Corning Costar Corporation, New York, NY, USA) and incubated for 24 h to attach. Cells were then treated with TKIs as follows: dasatinib (0.02–60 μM), erlotinib (0.32–40 μM), nilotinib (0.02–60 μM), regorafenib (0.02–4 μM), and sorafenib (0.02–4 μM) for 24 and 72 h, respectively. B(a)P (50 μM) was used as a positive control. After treatment, ZFL cells were trypsinized, collected, and centrifuged. Thirty μL of the cell suspension was mixed with 70 μL of 1% LMP agarose and placed on fully frosted slides pre-coated with 80 μL of 1% NMP agarose. Cells were then lysed for 1 h at 4 °C (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% Triton X-100, pH 10). DNA was subsequently denatured in electrophoresis buffer (1 mM EDTA, 300 mM NaOH, pH 13) for 20 min at 4 °C and slides were electrophoresed at 25 V (at 1 V/cm) for 20 min. Finally, nuclei were stained with GelRed (Biotium, Fremont, CA, USA). Images of 50 randomly selected nuclei per experimental point were acquired with the Eclipse 800 fluorescence microscope (Nikon, Tokyo, Japan) at 400× magnification using image analysis software (Comet Assay IV, Instem, UK). The statistical significance between control and treated groups was determined by one-way analysis of variance (ANOVA, Kruskal–Wallis) and Dunn’s multiple comparison test. * p < 0.05 was considered statistically significant.
Kološa K., Žegura B., Štampar M., Filipič M, & Novak M. (2023). Adverse Toxic Effects of Tyrosine Kinase Inhibitors on Non-Target Zebrafish Liver (ZFL) Cells. International Journal of Molecular Sciences, 24(4), 3894.
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Publication 2023
Buffers Cell Culture Techniques Cell Nucleus Cells Comet Assay Dasatinib DNA Breaks Edetic Acid Erlotinib Microscopy, Fluorescence neuro-oncological ventral antigen 2, human nilotinib regorafenib Sepharose Sodium Chloride Sorafenib Triton X-100 Tromethamine
4
Flow Cytometric Analysis of Apoptosis
TUNEL Assay Kit detects DNA fragmentation of apoptotic cells by marking DNA breaks using standard immunohistochemical techniques [23 (link)]. Thus, after treatment with the nanoparticles, the cells were fixed by incubation in 4% formaldehyde for 30 min. Cells were permeabilized using 0.2% Triton X-100 (in PBS) for 45 min at 37 °C to facilitate staining. All wells received a DNA marking solution (10 µL of reaction buffer, 0.75 µL of TdT enzyme, 8.0 µL of BrdUTP and 31.25 µL of dH2O) and incubated for 60 min at 37 °C. Afterwards, 1 mL of rinse buffer was added and the cells were centrifuged. The supernatant was removed and Alexafluor™488 was added. The nuclei were counterstained with propidium iodide (PI). Cells were stored in PBS at 4 °C until analysis and dUTP-labeled DNA was visualized by flow cytometry. Data acquisition and analysis were performed using a FACSCalibur flow cytometer (Becton-Dickinson, Rutherford, NJ, USA) equipped with CellQuest software (Joseph Trotter, Scripps Research Institute, La Jolla, CA, USA). A total of 10,000 events were acquired.
Hernandes E.P., Lazarin-Bidóia D., Bini R.D., Nakamura C.V., Cótica L.F, & de Oliveira Silva Lautenschlager S. (2023). Doxorubicin-Loaded Iron Oxide Nanoparticles Induce Oxidative Stress and Cell Cycle Arrest in Breast Cancer Cells. Antioxidants, 12(2), 237.
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Publication 2023
Apoptosis Biological Assay bromodeoxyuridine triphosphate Buffers Cell Nucleus Cells deoxyuridine triphosphate DNA, A-Form DNA Breaks DNA Fragmentation Enzymes Flow Cytometry Formaldehyde In Situ Nick-End Labeling Propidium Iodide Triton X-100
5
Sperm Chromatin Structure Assay
The sperm chromatin structure assay required a minimum of 10 μL semen samples with a concentration of ≥0.5 million/mL. Firstly, the frozen specimens were thawed in a 37°C water bath and re-suspended in TNE (Tris, NaCl and EDTA) buffer at the ratio of 1:9 (μL). Subsequently, the samples were treated with 400 μL of acid (pH1.20) for 30 s to denature DNA at the sites of strand breaks. Thirdly, acridine orange staining solution was added to stain the single-strand DNA breaks (presenting with red fluorescence) and double-strand DNA breaks (presenting with green fluorescence). At least 5,000 sperm cells were analysed per sample within 5 min using flow cytometry. The sperm fragmentation index was calculated as red fluorescence/(red and green fluorescence).
Sun T.C., Li D.M., Yu H., Song L.L., Jia Y.J., Lin L, & Zhou S.J. (2023). Bilateral varicocele leads to ferroptosis, pyroptosis and necroptosis of human spermatozoa and affects semen quality in infertile men. Frontiers in Cell and Developmental Biology, 11, 1091438.
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Publication 2023
Acids Acridine Orange Bath Biological Assay Buffers Cells Chromatin DNA Breaks DNA Breaks, Double-Stranded DNA Breaks, Single-Stranded Edetic Acid Flow Cytometry Fluorescence Freezing Plant Embryos Sodium Chloride Sperm Stains Tromethamine

Top products related to «DNA Breaks»

1
In situ cell death detection kit by Roche
Sourced in Germany, United States, Switzerland, China, United Kingdom, France, Canada, Belgium, Japan, Italy, Spain, Hungary, Australia
The In Situ Cell Death Detection Kit is a laboratory product designed for the detection of programmed cell death, or apoptosis, in cell samples. The kit utilizes a terminal deoxynucleotidyl transferase (TdT) to label DNA strand breaks, allowing for the visualization and quantification of cell death. The core function of this product is to provide researchers with a tool to study and analyze cell death processes.
2
Deadend fluorometric tunel system by Promega
Sourced in United States, Italy, Germany, United Kingdom, Australia, China, Poland
The DeadEnd Fluorometric TUNEL System is a laboratory equipment product that detects and quantifies apoptosis, or programmed cell death, in cells. The system utilizes terminal deoxynucleotidyl transferase (TdT) to label DNA strand breaks, which are characteristic of apoptotic cells. The labeled DNA is then detected using fluorescent dyes, allowing for the visualization and quantification of apoptotic cells.
3
Apoptag peroxidase in situ apoptosis detection kit by Merck Group
Sourced in United States, Germany, Canada, United Kingdom
The ApopTag Peroxidase In Situ Apoptosis Detection Kit is a laboratory tool designed to detect and visualize apoptosis, a programmed cell death process, in tissue samples. The kit utilizes enzymatic labeling and colorimetric detection to identify apoptotic cells.
4
Comet assay kit by Bio-Techne
Sourced in United States
The Comet Assay Kit is a laboratory tool used to assess DNA damage and repair at the single-cell level. It measures the extent of DNA fragmentation by evaluating the 'comet-like' tail formed during electrophoresis, which is proportional to the amount of DNA damage present in the cell.
5
Tunel assay kit by Roche
Sourced in United States, Switzerland, Germany, China, France, United Kingdom
The TUNEL assay kit is a laboratory tool used for the detection and quantification of DNA fragmentation, a hallmark of apoptosis or programmed cell death. The kit provides the necessary reagents and protocols to perform the TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick End Labeling) assay, which labels the free 3'-hydroxyl termini of fragmented DNA.
6
Apo brdu tunel assay kit by Thermo Fisher Scientific
Sourced in United States
The APO-BrdU TUNEL Assay Kit is a laboratory tool designed to detect and quantify apoptosis, a form of programmed cell death. The kit utilizes a terminal deoxynucleotidyl transferase (TdT) enzyme to incorporate bromodeoxyuridine (BrdU) into the fragmented DNA of apoptotic cells, which can then be detected using an anti-BrdU antibody.
7
In situ cell death detection kit by Merck Group
Sourced in United States, Germany
The In Situ Cell Death Detection Kit is a laboratory product designed to detect and analyze cell death in various biological samples. It provides a sensitive and reliable tool for researchers to assess apoptosis, necrosis, and other forms of programmed cell death during different experimental conditions.
8
Triton x 100 by Merck Group
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Triton X-100 is a non-ionic surfactant commonly used in various laboratory applications. It functions as a detergent and solubilizing agent, facilitating the solubilization and extraction of proteins and other biomolecules from biological samples.
9
Fluorescence microscope by Olympus
Sourced in Japan, United States, Germany, China, Italy, United Kingdom, Denmark, Switzerland, France
The Olympus Fluorescence Microscope is an optical microscope that uses fluorescence to visualize and analyze samples. It illuminates the specimen with light of a specific wavelength, causing fluorescent molecules within the sample to emit light at a different wavelength, which is then detected and displayed.
10
Dnase 1 by Roche
Sourced in United States, Switzerland, Germany, Japan, United Kingdom, France, Canada, Italy, Macao, China, Australia, Belgium, Israel, Sweden, Spain, Austria
DNase I is a lab equipment product that serves as an enzyme used for cleaving DNA molecules. It functions by catalyzing the hydrolytic cleavage of phosphodiester bonds in the DNA backbone, effectively breaking down DNA strands.

More about "DNA Breaks"

DNA double-strand breaks (DSBs) are critical disruptions in the continuity of the DNA molecule, which can be caused by various factors such as ionizing radiation, chemical agents, or cellular processes like DNA replication and repair.
These DNA lesions, if left unrepaired or misrepaired, can lead to genomic instability, cell death, and the development of genetic diseases, including cancer.
Understanding the mechanisms and effects of DNA breaks is crucial for advancing research in fields like molecular biology, genetics, and cancer biology.
Researchers often utilize specialized techniques and kits to detect and evaluate DNA breaks, such as the In Situ Cell Death Detection Kit, DeadEnd Fluorometric TUNEL System, and ApopTag Peroxidase In Situ Apoptosis Detection Kit.
These tools leverage techniques like the TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick End Labeling) assay, which labels DNA strand breaks, and the Comet assay, which measures DNA damage at the single-cell level.
In addition, the use of Triton X-100, a non-ionic detergent, can enhance the accessibility of DNA for these assays, while fluorescence microscopy provides a powerful visualization tool for analyzing DNA break patterns and quantifying their prevalence.
By effectively identifying and evaluating DNA break research protocols, scientists can accelerate scientific discovery and inform the development of new therapeutic strategies to address the challenges posed by these critical DNA lesions.
Platforms like PubCompare.ai, which leverage advanced AI-driven comparisons, can help researchers navigate the vast landscape of DNA break research and identify the most effective protocols and products to advance their work.