What does the comet assay detect?

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 is DNA damage measured?

DNA breaks and lesions may be detected by PCR or using agarose gel electrophoresis (7). ... Quantitative PCR (qPCR) has been performed to quantify the amount of DNA damage on both strands, as well as the kinetics of DNA damage removal in the mitochondrial DNA (mtDNA) of human and other organisms (7,9).

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 comet tail moment?

The tail moment is defined as the product of the tail length and the fraction of total DNA in the tail (Tail moment=tail length x % of DNA in the tail). This is calculated automatically by the computer software system as an average for the 50 cells selected for measurement.

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.

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.

What is neutral comet assay?

Comet assay under neutral conditions allows the detection of DNA double-strand breaks (DSB), considered to be the biologically relevant radiation-induced lesion.

What is tail length in comet assay?

Comet formation Comet assay or single cell gel electrophoresis assay detects single or double strand breaks measured at the individual cell level. ... In the comet structure, the undamaged DNA nucleoid part is referred to as the “head” and the trailing damaged DNA streak is referred to as the “tail” (Figure 43.4).

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.

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 does comet assay stand for?

• 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. It was first developed by Östling & Johansson in 1984 and later modified by Singh et al. in 1988.

Why to use the comet assay?

• The Comet Assay can be used to detect DNA damage caused by double strand breaks, single strand breaks, alkali labile sites, oxidative base damage, and DNA cross-linking with DNA or protein. The Comet Assay is also used to monitor DNA repair by living cells. 3

What equipment do I need for the comet assay?

• What equipment do I need? Fluorescence microscope & camera. In order to visualise your cells, you will need a microscope (upright or inverted style) with fluorescence. C-mount adapter. In order to connect a camera to your microscope, you will also need a C-mount adapter. ... Comet Assay IV scoring software. ... Horizontal electrophoresis tank. ...

What does comet assay mean?

• The Comet Assay, also called single cell gel electrophoresis (SCGE), is a sensitive and rapid technique for quantifying and analyzing DNA damage in individual cells. As such, this is one of the techniques used in the area of cancer research for the evaluation of genotoxicity and effectiveness of chemoprevention.