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P R O T O C O L S

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 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.


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.  

This assay has critically important applications in fields of toxicology ranging from aging and clinical investigations to genetic toxicology and molecular epidemiology.

Briefly, the single suspension of the cells of interest are suspended in low melting agarose and layered onto slides precoated with agarose. Lysis of the cells, under high salt concentration is then carried out to remove cellular proteins and liberate the damaged DNA. The liberated DNA is subjected to unwinding under alkaline / neutral conditions to allow DNA supercoils to relax and express DNA single strand breaks and alkali labile sites. Breaks may also be introduced at the site of damage by treatment with enzymes such as endonuclease and glycosylase and the fragment produced processed for comet assay. Electrophoresis is then carried out under neutral/highly alkaline (pH>13) conditions, which allows the broken ends to migrate under the effect of electric field, towards the anode. After neutralization, staining is done using fluorescent DNA dyes (e.g. Ethidium Bromide). The slides are then kept in a humidified slide chamber until they are scored. Hence, comet assay can be used to study various classes of DNA damage by controlling the conditions of the lysing and electrophoresis steps.

50-100 cells are counted per sample, by manual counting or using a software. Both qualitative and quantitative assessment of DNA damage is carried out. There are around 34 parameters monitored on the software, however the most frequently used are Tail DNA (%), Tail Length (mm), and Olive Tail Moment (Arbitrary units). 

The assay can be performed on a variety of samples which can be obtained as a single cell population e.g. peripheral blood lymphocytes, nasal and buccal epithelium from clinically or occupationally exposed human population and for in vitro studies on cell lines e.g. CHO, V79, mouse lymphoma or cultured human lymphocytes and bone marrow cells. Both DNA damage and repair studies can be conducted. Also a variety of information related to genetic toxicology, human epidemiology, patients undergoing radio/chemo- therapy, ageing and nutrition can be obtained. The assay has been used for environmental biomonitoring and has utilized earthworms, fishes, mollusks exposed to polluted environments.

The migrated DNA after neutralization is stained with appropriate dye [e.g. Ethidium Bromide, Propidium Iodide, YOYO-1, Hoescht 33258, DAPI and Acridine Orange] and visualized under a fluorescent microscope. For each treatment dose / individual  50-100 cells are counted using software and both qualitative and quantitative assessment of DNA damage is carried out.

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THE DETAILED PROTOCOLS FOR COMET ASSAY AND FOR APOPTOSIS
CAN BE DOWNLOADED AS PDF* 

COMET ASSAY PROTOCOL                          PROTOCOL FOR OXIDATIVE DNA DAMAGE           APOPTOSIS  PROTOCOL

PROTOCOL FOR DNA STRAND BREAKS                          SCHEME FOR VISUAL SCORING                APOPTOSIS PAPER

PROTOCOL FOR COMET ASSAY IN SPERM                                 SPERM COMET PAPER            APOPTOSIS PHOTOGRAPH

GELBOND COMET ASSAY                             

*THESE PDF FILES ARE ONLY FOR PERSONAL USE AND NOT FOR DISTRIBUTION OR COMMERCIAL PURPOSES