What is alkali labile sites?
Alkali labile sites (ALS) are defined as native DNA motifs highly sensitive toincubation in alkali solutions which produce portions of single-stranded DNA (ss-DNA)identified by alkaline single-cell gel electrophoresis or alkaline elution assays.
Is comet assay reliable?
Introduction. Testing of chemicals for genotoxicity requires methods that are sensitive and specific, preferably economical and quick, and reliable. The alkaline (pH > 13) comet assay (single-cell gel electrophoresis) meets most of these requirements, but is regarded by some as unreliable.
What does comet assay measure?
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 conduct 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.
What is the meaning of Comet in comet assay?
The single cell gel electrophoresis assay (SCGE, also known as comet assay) is an uncomplicated and sensitive technique for the detection of DNA damage at the level of the individual eukaryotic cell. The term “comet” refers to the pattern of DNA migration through the electrophoresis gel, which often resembles a comet.
Why is alkaline comet assay?
The alkaline comet assay is capable of detecting DNA double-strand breaks, single-strand breaks, alkali-labile sites, DNA-DNA/DNA-protein cross-linking, and incomplete excision repair sites.
What is alkaline elution?
Abstract. The alkaline elution technique, developed by Kohn and coworkers (Kohn and Ewig, 1973; Kohn et al., 1976; Kohn, 1979), provides a sensitive measurement of DNA single-strand size distribution in mammalian cells and represents an advanced alternative to the technique of alkaline sucrose gradient sedimentation.
How do you store a comet assay slide?
To preserve comet assay slides after staining you can drain slides, keep them for 20 min in cold 100% ethanol or cold 100% methanol for dehydration. Air dry the slides and place them in an oven at 50 C for 30 min. Store in a dry area.
How many cells does a comet assay have?
Thus by counting a representative sample of ∼ 100–300 cells per tissue it is possible to arrive at the average percentage of DNA damage accumulated in a particular tissue due to genotoxic stress. The comet assay is also referred to as SCGE assay.
What is the sensitivity of the comet assay?
The sensitivity of the assay depends on its calibration and there are some variations between laboratories, although substantial efforts have been made to standardize the assay. It is generally accepted that the frequency of DNA damage detectable with the Comet assay is from 0.06 to 3 breaks per 109 Da of genomic DNA.
What is the use of ctcomet assay?
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.
Can comet assay be used to measure sperm DNA damage?
The level of sperm DNA damage measured by the comet assay is closely associated to other direct method such as TUNEL assay (Shamsi et al., 2008 ). The comet assay can be used to measure DNA damage in a variety of cell types, including sperm. The assay requires only a few of cells to perform the procedure.
What is the target tissue for in vivo comet assay?
Although in principle any tissue can be used for in vivo comet assay analysis, high blood circulating organs such as liver, kidney, and spleen are the target organ tissues that are analyzed. Also, if the route of administration is oral, then the site of contact or exposed tissue such as stomach mucosal cells are commonly used for comet analyses.