An optimized comet-based in vitro DNA repair assay to assess base and nucleotide excision repair activity.

Nature Protocols
Sona VodenkovaSabine A S Langie

Abstract

This optimized protocol (including links to instruction videos) describes a comet-based in vitro DNA repair assay that is relatively simple, versatile, and inexpensive, enabling the detection of base and nucleotide excision repair activity. Protein extracts from samples are incubated with agarose-embedded substrate nucleoids ('naked' supercoiled DNA) containing specifically induced DNA lesions (e.g., resulting from oxidation, UVC radiation or benzo[a]pyrene-diol epoxide treatment). DNA incisions produced during the incubation reaction are quantified as strand breaks after electrophoresis, reflecting the extract's incision activity. The method has been applied in cell culture model systems, human biomonitoring and clinical investigations, and animal studies, using isolated blood cells and various solid tissues. Once extracts and substrates are prepared, the assay can be completed within 2 d.

References

Mar 1, 1988·Experimental Cell Research·N P SinghE L Schneider
Jun 1, 1971·Journal of the Neurological Sciences·K G Kjellin
Aug 30, 1984·Biochemical and Biophysical Research Communications·O Ostling, K J Johanson
Jul 22, 1997·Proceedings of the National Academy of Sciences of the United States of America·T Roldán-ArjonaT Lindahl
Aug 5, 2000·Free Radical Biology & Medicine·R M ElliottD B Archer
Mar 29, 2003·Carcinogenesis·Andrew R CollinsRachel Melvin
Sep 4, 2003·Journal of the National Cancer Institute·Tamar Paz-ElizurZvi Livneh
Aug 4, 2004·Mutation Research·Mária DusinskáSoterios Kyrtopoulos
Aug 4, 2004·Mutation Research·Mária DusinskáAndrew Collins
Aug 10, 2007·Environmental Health Perspectives·Elvira Vaclavik BräunerSteffen Loft
Oct 11, 2007·The British Journal of Nutrition·Serena GuarnieriPeter Møller
Feb 20, 2008·Mutagenesis·Andrew R CollinsRudolf Stetina
May 23, 2008·Mutation Research·Pernille Høgh DanielsenSteffen Loft
Jul 8, 2008·Mutation Research·Tomasz SliwinskiJanusz Blasiak
Nov 11, 2008·European Journal of Pharmacology·João P SilvaOlga P Coutinho
Feb 11, 2009·Journal of Wildlife Diseases·Yannick RomanMichel Saint Jalme
May 30, 2009·Environmental Health Perspectives·Janne K FolkmannPeter Møller
Jun 9, 2009·Free Radical Biology & Medicine·Lone MikkelsenPeter Møller
Aug 20, 2009·Clinical Cancer Research : an Official Journal of the American Association for Cancer Research·Mercedes HerreraFelix Bonilla
Oct 22, 2009·Mutagenesis·Kim Vande LoockMicheline Kirsch-Volders
Nov 3, 2009·The British Journal of Nutrition·Sabine A S LangieFrederik J van Schooten
Nov 18, 2009·Mutagenesis·Nejla GüngörFrederik J van Schooten
Dec 18, 2009·Mutation Research·Sabine A S LangieRoger W L Godschalk
Mar 2, 2010·Toxicology Letters·Sergey ShaposhnikovAndrew R Collins
May 22, 2010·Journal of Agricultural and Food Chemistry·Alice A RamosAndrew R Collins
Aug 18, 2010·Mutagenesis·Patrizia RisoMarisa Porrini
Sep 11, 2010·Biochemical and Biophysical Research Communications·E van DykP J Pretorius

❮ Previous
Next ❯

Citations

Jul 17, 2021·Mutation Research. Genetic Toxicology and Environmental Mutagenesis·Eliana E OcolotobicheAlba Mabel Güerci
Oct 6, 2021·Archives of Toxicology·Ezgi Eyluel BankogluHelga Stopper
Oct 16, 2021·Chemical Communications : Chem Comm·Wei XueMeng Liu

❮ Previous
Next ❯

Methods Mentioned

BETA
electrophoresis
fluorescence microscopy
biopsy
protein
Protein Assay
Assay
PCR
density gradient centrifugation
irradiate

Software Mentioned

ImageJ
CaspLab
Comet Assay IV
Metafer
OpenComet

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.