HELIX3
INC.

The Comet Assay

Introduction

The alkaline version of the comet assay was developed to measure DNA damage (or its repair) in virtually any cell type.  Prior to the comet assay, tests used to measure the potential health risk of exposures to food additives, pharmaceuticals, chemicals, and/or environmental factors were limited to insensitive and often non-specific tests that were inappropriate for assessing the true risk of exposures.  Today, the comet assay can be used to measure the very low levels of DNA damage and/or repair inhibition that may lead to carcinogenesis and other health risks.

Important Information

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Description

The comet or single cell gel (SCG) assay measures DNA damage (e.g. strand breaks, DNA adducts, excision repair sites, and crosslinks) at the single cell level.  Without the necessity for special cell isolation techniques, a small amount of cells is embedded in an agarose gel matrix on a microscope slide and lysed to remove the cell membrane and most of the cellular proteins.

Types of DNA Damage Detected

Strand Breaks

DNA Adducts

Crosslinks

Oxidative Damage

DNA migration is measured by image analysis

Cells are embedded in agarose on a slide

Lysis removes cell membranes leaving only nuclear DNA on slides

Alkaline (pH>13) buffer denatures DNA to express strand breaks

DNA pieces are migrated away from the nucleus

Once the cells are lysed, an electric current is applied to the nuclear DNA causing DNA fragements to migrate away from the nucleus or the head of the comet at a rate and distance directly proportional to the size of the fragment.  The pixel intensity of the migrated fragments can then be measured to determine the DNA migration levels as an indication of damage and/or repair in a population of cells.

Benefits

As a tool for measuring genotoxicity, the comet assay offers unique flexibility by being applicable to virtually any cell type (in vitro or in vivo).  In knowledgable hands, the assay is a powerful predictor of carcinogenicity and other health effects where standard tests have proven to be ineffective.

Faster and more sensitive than standard assays, the comet assay is the most cost effect method for the detection of health risks during drug and chemical development, efficacy testing, environmental monitoring, and human exposure analysis.  And since the comet assay can be incorporated into standard assays, it can reduce the time and expense associated with testing while maximizing the information provided to determine risk.