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Comet Assay Abstract Search Software (C)- A software that keeps you updated with the comet assay literature - for details contact  alokdhawan@comet.itrcindia.org

I N T R O D U C T I O N  


Several man-made chemicals find their way into the environment and pose health risk to human population. These chemicals have been found to interact with the vital tissue macromolecules regulating the cellular functions leading to long lasting health disorders. Acute and chronic exposure to several of these environmental chemicals such as pesticide, metals, polycyclic aromatic hydrocarbons (PAHs), solvents etc. have been shown to produce marked toxicity at the target sites. Some of these chemicals affect the DNA, which is the carrier of inherited information and any gross change in its structure potentates serious biological changes. Hence there is a need to test the chemicals for their genotoxic potential before being released into the environment. The conventional methods for evaluating genetic damage include chromosomal aberration, micronucleus assay, sister chromatid exchanges. However these are time consuming, resource intensive and require proliferating cell population. Hence newer and more sensitive test systems have now been introduced for assessing the genotoxicity of chemicals.

The single cell gel electrophoresis or comet assay is one such state-of-the-art technique for quantitating DNA damage and repair in vivo and in vitro in any eukaryotic cell and some prokaryotic cells. This technique is rapid, non-invasive, sensitive, visual and inexpensive as compared to the conventional techniques and is a powerful tool to study factors modifying mutagenicity and carcinogenicity. It has rapidly gained importance in the fields of genetic toxicology and human biomonitoring.

WHAT COMET ASSAY MEASURES:

Comet assay measures, double strand breaks (DSBs), single strand breaks (SSBs), alkali labile sites, oxidative DNA base damage, DNA-DNA/DNA-protein/DNA-Drug crosslinking and DNA repair.

 

PRINCIPLE:

The assay works upon the principle that strand breakage of the supercoiled duplex DNA leads to the reduction of the size of the large molecule and these strands can be stretched out by electrophoresis. Also, under highly alkaline conditions there is denaturation, unwinding of the duplex DNA and expression of alkali labile sites as single strand breaks. Comets form as the broken ends of the negatively charged DNA molecule become free to migrate in the electric field towards the anode. Two principles in the formation of the comet are :

1.      DNA migration is a function of both size and the number of broken ends of the DNA

2.      Tail length increases with damage initially and then reaches a maximum that is dependent on the electrophoretic conditions, not the size of fragments.

 HISTORY:

The comet assay and microgel electrophoresis (MGE) were first introduced by Ostling and Johanson in 1984. This was a neutral assay in which the lysis and electrophoresis were done under neutral conditions.
Staining was done with acridine orange. The image obtained looked like a “comet” with a distinct head, comprising of intact DNA and a tail, consisting of damaged or broken pieces of DNA hence the name “Comet” Assay was given. The extent of DNA liberated from the head of the comet was the function of the dose of irradiation. However, in this procedure, only double strand breaks could be analyzed.

The above neutral assay was modified by two groups, Singh and co-workers (1988) and Olive et al (1989). Singh et al used microgels, involving electrophoresis under highly alkaline conditions (ph>13). This enabled the DNA supercoils to get relaxed and unwind, which are then pulled out during application of electric-current which made possible the detection of single strand breaks in DNA and alkali labile sites expressed as frank single strand breaks in individual cells. This method was developed to measure low levels of strand breaks with high sensitivity.


Olive and co-workers conducted the electrophoresis under neutral or mild alkaline  (pH=12.3) to detect single stranded breaks. This method was optimized to detect a subpopulation of cells with varying sensitivity to drug or radiation. The technique of Singh et al was found to be one or two orders of magnitude more sensitive than the other techniques.


Since then a number of advancements have greatly increased the flexibility and utility of this technique for detecting various forms of DNA damage (e.g., single- and double-strand breaks, oxidative DNA base damage, and DNA-DNA/DNA-protein/DNA-Drug crosslinking) and DNA repair in virtually any eukaryotic cell. 

 METHODOLOGY : 

 FOR DETAILED PROTOCOL CLICK HERE

APPLICATIONS:

Major applications of the Comet assay are in the following areas:

 

  • Genetic toxicology (DNA damage)
               
    In vivo & in vitro evaluation of genotoxic chemicals

  • DNA damage:
      
             SSB’s, DNA crosslinking, alkali labile sites

  • DNA repair:
      
             Strand break repair
               
    Excision repair

  • Eco-toxicology: the assay has been used to monitor soil and aquatic toxicology

  • Nutrition

  • Bio-monitoring genotoxicity

  • Environmental biomonitoring
               
    Evaluation of genotoxic pollutants from hazardous waste sites

  • Hypoxia assessment

  • Human epidemiology
               
    For assessing levels of DNA damage in occupationally, clinically and environmentally exposed individuals or in evaluating the differences in DNA repair competency among control and exposed individuals.
      
             (a) Sperm bank
      
             (b) Blood bank
               
    (c) Monitoring Radio- and chemo- therapy in cancer patients

  • Miscellaneous

FOR REFERENCES ON VARIOUS APPLICATION CLICK HERE

ADVANTAGES:

There are many advantages of the comet assay, some of which are:

  • it is a non-invasive technique

  • it requires <10,000 cells and collection of data at the level of the individual cell, allowing for more robust types of statistical analyses

  • counting of 50-100 cells per individual / treatment group, through a computerised image analysis software gives a robust statistics 

  • that virtually any eukaryotic cell population is amenable to analysis

  • its sensitivity (1 break in 1010 daltons) for detecting DNA damage and repair

  • results obtained in a few hours compared to conventional cytogenetics techniques which take a few days

  • single strand breaks (SSB’s) and alkali labile lesions (capable of being transformed into SSB’s under alkaline conditions) in the DNA of individual cells can be assessed

  • only few microlitres of blood (5-10ml), nasal & buccal mucosal cells, epithelial cells, male germ cells, fine needle biopsy, etc. required human studies.

 


Last updated on: April 3, 2006
Copyright © 2008 Indian Institute of Toxicology Research, India
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