Comet assay
Encyclopedia
The Single Cell Gel Electrophoresis assay (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 described by Singh et al. in 1988. It has since gained in popularity as a standard technique for evaluation of DNA damage/repair, biomonitoring and genotoxicity testing. It involves the encapsulation of cells in a low-melting-point agarose suspension, lysis of the cells in neutral or alkaline (pH>13) conditions, and electrophoresis of the suspended lysed cells. This is followed by visual analysis with staining of DNA and calculating fluorescence to determine the extent of DNA damage. This can be performed by manual scoring or automatically by an imaging software.
cell culture
or from an in vivo
test subject is dispersed into individual cells and suspended in molten low-melting-point agarose at 37°C. This mono-suspension is cast on a microscope slide
. A glass cover slip
is held at an angle and the mono-suspension applied to the point of contact between the coverslip and the slide. As the coverslip is lowered onto the slide the molten agarose spreads to form a thin layer. The agarose is gelled at 4°C and the coverslip removed.
The agarose forms a matrix of carbohydrate
fibres that encapsulate the cells, anchoring them in place. The agarose is considered to be osmotic-neutral, therefore solution
s can penetrate the gel and affect the cells without cells shifting position.
In an in vitro study the cells would be exposed to a test agent - typically UV light, ionising radiation, or a genotoxic
chemical - to induce DNA damage in the encapsulated cells. For calibration
, hydrogen peroxide
is usually used to provide a standardized level of DNA damage.
. The lysis solution often used in the comet assay consists of a highly concentrated aqueous salt
(often, common table salt can be used) and a detergent
(such as Triton X-100 or sarcosinate
). The pH
of the lysis solution can be adjusted (usually between neutral and alkaline pH) depending upon the type of damage the researcher is investigating.
The aqueous salt disrupts protein
s and their bonding patterns within the cell as well as disrupting the RNA
content of the cell. The detergent dissolves the cellular membranes. Through the action of the lysis solution the cells are destroyed. All proteins, RNA, membranes and cytoplasm
ic and nucleoplasm
ic constituents are disrupted and diffuse into the agarose matrix. Only the DNA
of the cell remains, and unravels to fill the cavity in the agarose that the whole cell formerly filled. This structure is called nucleoid (a general term for a structure in which DNA is concentrated).
to remove all salts and immersed in a second solution - an electrophoresis
solution. Again this solution can have its pH adjusted depending upon the type of damage that is being investigated.
The slides are left for ~20 minutes in the electrophoresis solution prior to an electric field
being applied. In alkaline conditions the DNA double helix is denature
d and the nucleoid becomes single stranded.
An electric field is applied (typically 1 V
/cm) for ~20 minutes. The slides are then neutralised to pH 7, stained with a DNA-specific fluorescent stain and analysed using a microscope
with an attached CCD (charge-coupled device
- essentially a digital camera
) that is connected to a computer with image analysis
software.
The image analysis measures the overall intensity of the fluorescence for the whole nucleoid and the fluorescence of the migrated DNA and compares the two signals. The stronger the signal from the migrated DNA the more damage there is present. The overall structure resembles a comet
(hence "comet assay") with a circular head corresponding to the undamaged DNA that remains in the cavity and a tail of damaged DNA. The brighter and longer the tail, the higher the level of damage.
The comet assay is a versatile technique for detecting damage and with adjustments to the protocol can be used to quantify the presence of a wide variety of DNA altering lesions (damage). The damage usually detected are single strand breaks and double strand breaks. It is sometimes stated that alkaline conditions and complete denaturating of the DNA is necessary to detect single strand breaks. However this is not true, both single- and double strand breaks are also detected in neutral conditions. In alkaline conditions, however, additional DNA structures are detected as DNA damage: AP sites (abasic sites missing either a pyrimidine
or purine
nucleotide
) and sites where excision repair
is taking place.
The comet assay is an extremely sensitive DNA damage assay. This sensitivity needs to be handled carefully as it is also vulnerable to physical changes which can affect the reproducibility of results. Essentially, anything that can cause DNA damage or denaturation except the factor(s) being researched is to be avoided. The most common form of the assay is the alkaline version although there is as yet no definitive alkaline assay protocol. Due to its simple and inexpensive setup, it can be used in conditions where more complex assays are not available.
in sperm cells. The degree of DNA fragmentation can predict outcomes for in vitro fertilization.
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
. It was first described by Singh et al. in 1988. It has since gained in popularity as a standard technique for evaluation of DNA damage/repair, biomonitoring and genotoxicity testing. It involves the encapsulation of cells in a low-melting-point agarose suspension, lysis of the cells in neutral or alkaline (pH>13) conditions, and electrophoresis of the suspended lysed cells. This is followed by visual analysis with staining of DNA and calculating fluorescence to determine the extent of DNA damage. This can be performed by manual scoring or automatically by an imaging software.
Encapsulation
A sample of cells, either derived from an in vitroIn vitro
In vitro refers to studies in experimental biology that are conducted using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis than can be done with whole organisms. Colloquially, these experiments...
cell culture
Cell culture
Cell culture is the complex process by which cells are grown under controlled conditions. In practice, the term "cell culture" has come to refer to the culturing of cells derived from singlecellular eukaryotes, especially animal cells. However, there are also cultures of plants, fungi and microbes,...
or from an in vivo
In vivo
In vivo is experimentation using a whole, living organism as opposed to a partial or dead organism, or an in vitro controlled environment. Animal testing and clinical trials are two forms of in vivo research...
test subject is dispersed into individual cells and suspended in molten low-melting-point agarose at 37°C. This mono-suspension is cast on a microscope slide
Microscope slide
A microscope slide is a thin flat piece of glass, typically 75 by 25 mm and about 1 mm thick, used to hold objects for examination under a microscope. Typically the object is placed or secured on the slide, and then both are inserted together in the microscope for viewing...
. A glass cover slip
Cover slip
A cover slip or cover glass is a thin flat piece of transparent material, usually square or rectangular, about 20 mm wide and a fraction of a millimetre thick, that is placed over objects for viewing with a microscope...
is held at an angle and the mono-suspension applied to the point of contact between the coverslip and the slide. As the coverslip is lowered onto the slide the molten agarose spreads to form a thin layer. The agarose is gelled at 4°C and the coverslip removed.
The agarose forms a matrix of carbohydrate
Carbohydrate
A carbohydrate is an organic compound with the empirical formula ; that is, consists only of carbon, hydrogen, and oxygen, with a hydrogen:oxygen atom ratio of 2:1 . However, there are exceptions to this. One common example would be deoxyribose, a component of DNA, which has the empirical...
fibres that encapsulate the cells, anchoring them in place. The agarose is considered to be osmotic-neutral, therefore solution
Solution
In chemistry, a solution is a homogeneous mixture composed of only one phase. In such a mixture, a solute is dissolved in another substance, known as a solvent. The solvent does the dissolving.- Types of solutions :...
s can penetrate the gel and affect the cells without cells shifting position.
In an in vitro study the cells would be exposed to a test agent - typically UV light, ionising radiation, or a genotoxic
Genotoxic
In genetics, genotoxicity describes a deleterious action on a cell's genetic material affecting its integrity. This includes both certain chemical compounds and certain types of radiation....
chemical - to induce DNA damage in the encapsulated cells. For calibration
Calibration
Calibration is a comparison between measurements – one of known magnitude or correctness made or set with one device and another measurement made in as similar a way as possible with a second device....
, hydrogen peroxide
Hydrogen peroxide
Hydrogen peroxide is the simplest peroxide and an oxidizer. Hydrogen peroxide is a clear liquid, slightly more viscous than water. In dilute solution, it appears colorless. With its oxidizing properties, hydrogen peroxide is often used as a bleach or cleaning agent...
is usually used to provide a standardized level of DNA damage.
Lysis
The slides are then immersed in a solution that cause the cells to lyseLysis
Lysis refers to the breaking down of a cell, often by viral, enzymic, or osmotic mechanisms that compromise its integrity. A fluid containing the contents of lysed cells is called a "lysate"....
. The lysis solution often used in the comet assay consists of a highly concentrated aqueous salt
Salt
In chemistry, salts are ionic compounds that result from the neutralization reaction of an acid and a base. They are composed of cations and anions so that the product is electrically neutral...
(often, common table salt can be used) and a detergent
Detergent
A detergent is a surfactant or a mixture of surfactants with "cleaning properties in dilute solutions." In common usage, "detergent" refers to alkylbenzenesulfonates, a family of compounds that are similar to soap but are less affected by hard water...
(such as Triton X-100 or sarcosinate
Sodium lauroyl sarcosinate
Sodium lauroyl sarcosinate , also known as sarkosyl, is an ionic surfactant derived from sarcosine, used as a foaming and cleansing agent in shampoo, shaving foam and foam wash products....
). The pH
PH
In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...
of the lysis solution can be adjusted (usually between neutral and alkaline pH) depending upon the type of damage the researcher is investigating.
The aqueous salt disrupts protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
s and their bonding patterns within the cell as well as disrupting the RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
content of the cell. The detergent dissolves the cellular membranes. Through the action of the lysis solution the cells are destroyed. All proteins, RNA, membranes and cytoplasm
Cytoplasm
The cytoplasm is a small gel-like substance residing between the cell membrane holding all the cell's internal sub-structures , except for the nucleus. All the contents of the cells of prokaryote organisms are contained within the cytoplasm...
ic and nucleoplasm
Nucleoplasm
Similar to the cytoplasm of a cell, the nucleus contains nucleoplasm or karyoplasm. The nucleoplasm is one of the types of protoplasm, and it is enveloped by the nuclear membrane or nuclear envelope. The nucleoplasm is a highly viscous liquid that surrounds the chromosomes and nucleoli...
ic constituents are disrupted and diffuse into the agarose matrix. Only the DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
of the cell remains, and unravels to fill the cavity in the agarose that the whole cell formerly filled. This structure is called nucleoid (a general term for a structure in which DNA is concentrated).
Electrophoresis
After lysis of the cells (typically 1 to 2 hours at 4°C) the slides are washed in distilled waterDistilled water
Distilled water is water that has many of its impurities removed through distillation. Distillation involves boiling the water and then condensing the steam into a clean container.-History:...
to remove all salts and immersed in a second solution - an electrophoresis
Electrophoresis
Electrophoresis, also called cataphoresis, is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field. This electrokinetic phenomenon was observed for the first time in 1807 by Reuss , who noticed that the application of a constant electric...
solution. Again this solution can have its pH adjusted depending upon the type of damage that is being investigated.
The slides are left for ~20 minutes in the electrophoresis solution prior to an electric field
Electric field
In physics, an electric field surrounds electrically charged particles and time-varying magnetic fields. The electric field depicts the force exerted on other electrically charged objects by the electrically charged particle the field is surrounding...
being applied. In alkaline conditions the DNA double helix is denature
Denaturation (biochemistry)
Denaturation is a process in which proteins or nucleic acids lose their tertiary structure and secondary structure by application of some external stress or compound, such as a strong acid or base, a concentrated inorganic salt, an organic solvent , or heat...
d and the nucleoid becomes single stranded.
An electric field is applied (typically 1 V
Volt
The volt is the SI derived unit for electric potential, electric potential difference, and electromotive force. The volt is named in honor of the Italian physicist Alessandro Volta , who invented the voltaic pile, possibly the first chemical battery.- Definition :A single volt is defined as the...
/cm) for ~20 minutes. The slides are then neutralised to pH 7, stained with a DNA-specific fluorescent stain and analysed using a microscope
Microscope
A microscope is an instrument used to see objects that are too small for the naked eye. The science of investigating small objects using such an instrument is called microscopy...
with an attached CCD (charge-coupled device
Charge-coupled device
A charge-coupled device is a device for the movement of electrical charge, usually from within the device to an area where the charge can be manipulated, for example conversion into a digital value. This is achieved by "shifting" the signals between stages within the device one at a time...
- essentially a digital camera
Digital camera
A digital camera is a camera that takes video or still photographs, or both, digitally by recording images via an electronic image sensor. It is the main device used in the field of digital photography...
) that is connected to a computer with image analysis
Image analysis
Image analysis is the extraction of meaningful information from images; mainly from digital images by means of digital image processing techniques...
software.
Imaging and Analysis Software
- Commercial software - listed by Comet Assay Interest Group
- CaspLab (CaspLab.com) - GPL software.
- Cometscore - free (freeware) program for semi-automatic scoring of the comets, compatible with XP and Vista.
Commercially Available Kits and Reagents
CometAssay Kits, Reagents, Control Cells and Electrophoresis Systems are available from Trevigen, Inc.. Trevigen has developed a complete comet assay system which includes CometAssay kits, CometSlides, CometAssay control cells and a specialized electrophoresis unit. The CometAssay electrophoresis unit maintains constant buffer temperature and optimal buffer height. Specially designed trays accommodate 2, 20 and 96 well slides and maintain correct slide orientation during electrophoresis.Background
The concept underlying the SCGE assay is that undamaged DNA retains a highly organized association with matrix proteins in the nucleus. When damaged, this organization is disrupted. The individual strands of DNA lose their compact structure and relax, expanding out of the cavity into the agarose. When the electric field is applied the DNA, which has an overall negative charge, it is drawn towards the anode, which is positively charged. Undamaged DNA strands are too large and do not leave the cavity, whereas the smaller the fragments, the farther they are free to move in a given period of time. Therefore, the amount of DNA that leaves the cavity is a measure of the amount of DNA damage in the cell.The image analysis measures the overall intensity of the fluorescence for the whole nucleoid and the fluorescence of the migrated DNA and compares the two signals. The stronger the signal from the migrated DNA the more damage there is present. The overall structure resembles a comet
Comet
A comet is an icy small Solar System body that, when close enough to the Sun, displays a visible coma and sometimes also a tail. These phenomena are both due to the effects of solar radiation and the solar wind upon the nucleus of the comet...
(hence "comet assay") with a circular head corresponding to the undamaged DNA that remains in the cavity and a tail of damaged DNA. The brighter and longer the tail, the higher the level of damage.
The comet assay is a versatile technique for detecting damage and with adjustments to the protocol can be used to quantify the presence of a wide variety of DNA altering lesions (damage). The damage usually detected are single strand breaks and double strand breaks. It is sometimes stated that alkaline conditions and complete denaturating of the DNA is necessary to detect single strand breaks. However this is not true, both single- and double strand breaks are also detected in neutral conditions. In alkaline conditions, however, additional DNA structures are detected as DNA damage: AP sites (abasic sites missing either a pyrimidine
Pyrimidine
Pyrimidine is a heterocyclic aromatic organic compound similar to benzene and pyridine, containing two nitrogen atoms at positions 1 and 3 of the six-member ring...
or purine
Purine
A purine is a heterocyclic aromatic organic compound, consisting of a pyrimidine ring fused to an imidazole ring. Purines, including substituted purines and their tautomers, are the most widely distributed kind of nitrogen-containing heterocycle in nature....
nucleotide
Nucleotide
Nucleotides are molecules that, when joined together, make up the structural units of RNA and DNA. In addition, nucleotides participate in cellular signaling , and are incorporated into important cofactors of enzymatic reactions...
) and sites where excision repair
Excision repair
Excision repair is a term applied to several DNA repair mechanisms. They remove the damaged nucleotides and are able to determine the correct sequence from the complementary strand of DNA.Specific mechanisms include:...
is taking place.
The comet assay is an extremely sensitive DNA damage assay. This sensitivity needs to be handled carefully as it is also vulnerable to physical changes which can affect the reproducibility of results. Essentially, anything that can cause DNA damage or denaturation except the factor(s) being researched is to be avoided. The most common form of the assay is the alkaline version although there is as yet no definitive alkaline assay protocol. Due to its simple and inexpensive setup, it can be used in conditions where more complex assays are not available.
Sperm DNA fragmentation
A Comet assay can determine the degree of DNA fragmentationDNA fragmentation
DNA fragmentation is the separation or breaking of DNA strands into pieces. It can be intentional by laboratory personnel or the cells, or it can be spontaneous.-Intentional:Restriction digest is the intentional laboratory breaking of DNA strands....
in sperm cells. The degree of DNA fragmentation can predict outcomes for in vitro fertilization.