How is oxidative DNA damage measured?
A wide variety of tools are available to measure DNA damage, in particular for the nuclear genome. Some of the most widely used tools to measure oxidative DNA damage are the comet assay, PCR-based assays, immunoassays, and mass spectrometry-based approaches.
What type of DNA is most susceptible to oxidative damage?
As described in detail by Burrows et al., 8-oxo-2′-deoxyguanosine (8-oxo-dG) is the most common oxidative lesion observed in duplex DNA because guanine has a lower one-electron reduction potential than the other nucleosides in DNA.
How does oxidative stress damage DNA?
Oxidative damage, produced by intracellular ROS, results in DNA base modifications, single- and double-strand breaks, and the formation of apurinic/apyrimidinic lesions, many of which are toxic and/or mutagenic (7).
What is DNA comet assay?
The comet assay (single-cell gel electrophoresis) is a simple method for measuring deoxyribonucleic acid (DNA) strand breaks in eukaryotic cells. Cells embedded in agarose on a microscope slide are lysed with detergent and high salt to form nucleoids containing supercoiled loops of DNA linked to the nuclear matrix.
How do you do a comet assay?
Comet assay utilizes single cells to measure DNA damage. First, cells are embedded into agarose and then placed onto a slide. The slide is then immersed into lysis solution to break open the cell membrane. After the cells are lysed, DNA is denatured under neutral or alkaline conditions and run through electrophoresis.
How is DNA damage measured?
Breaks in DNA reduce the molecular weight of a single DNA strand, and this may be caused by physical, chemical or enzymatic reagents (6). DNA breaks and lesions may be detected by PCR or using agarose gel electrophoresis (7). PCR is one of the most frequently used techniques for detecting DNA damage (7).
How does oxidative damage occur?
Oxidative stress can occur when there is an imbalance of free radicals and antioxidants in the body. The body’s cells produce free radicals during normal metabolic processes. However, cells also produce antioxidants that neutralize these free radicals.
What is the primary source of oxidative DNA damage in cells?
To avoid the deleterious consequences of damage accumulation, multiple DNA repair pathways have evolved, each associated with specific classes of lesions (5–8). The major source of endogenous DNA damage is reactive oxygen species (ROS) generated from normal cellular metabolism (9,10).
What does the comet assay detect?
Comet assay is a microgel electrophoresis technique, which detects DNA damage and repair in individual cells. The assay measures DNA damage (i.e., strand breaks, DNA adducts, excision repair sites, and cross-links) at the single-cell level.
How do you test for DNA damage?
The comet assay or single-cell gel (SCG) test is a microgel electrophoresis technique that measures DNA damage at the level of single cells. A small number of cells suspended in a thin agarose gel on a microscope slide is lysed, electrophoresed, and stained with a fluorescent DNA binding dye.