Comet assay
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206339492
2008-04-17T21:31:31Z
CmdrObot
1079367
sp (2): an low→a low, in vitro→''in vitro''
The '''S'''ingle '''C'''ell '''G'''el '''E'''lectrophoresis 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 (biology)|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.
==Experimental procedure==
===Encapsulation===
A sample of cells, either derived from an ''[[in vitro]]'' [[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.
===Lysis===
The slides are then immersed in a solution that cause the cells to [[lyse]]. 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 [[Sodium lauroyl sarcosinate|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 membrane]]s. 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).
===Electrophoresis===
After lysis of the cells (typically 1 to 2 hours at 4°C) the slides are washed in [[distilled water]] 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 [[volt|V]]/[[centimeter|cm]]) for ~20 minutes. The slides are then neutralised to pH 7, stained with a [[Staining#Basic biological stains|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.
====Imaging software====
* [http://casplab.com CASPLab] - GPL software.
* [http://cometassay.com/imaging_software.htm Commercial software] - listed by Comet Assay Interest Group
==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 is drawn towards the [[anode]]. 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]] (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<ref>Theoretical and practical limitations to the assay are discussed e.g. in Klaude ''et al.'' (1996) and Collins ''et al.'' (1997).</ref>. 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.
==Footnotes==
<references/>
==References==
* {{aut|Avishai, Nanthawan; Rabinowitz, Claudette; Moiseeva, Elisabeth & Rinkevich, Baruch}} (2002): Genotoxicity of the Kishon River, Israel: the application of an ''in vitro'' cellular assay. ''[[Mutation Research]]'' '''518'''(1): 21–37. <small>{{doi|10.1016/S1383-5718(02)00069-4}}</small> (HTML abstract)
* {{aut|Collins, A.R.; Dobson, V.L.; Dusinska, M.; Kennedy, G. & Stetina, R.}} (1997): The comet assay: what can it really tell us? ''Mutation Research'' '''375'''(2): 183-193. <small>{{doi|10.1016/S0027-5107(97)00013-4}}</small> (HTML abstract)
* {{aut|Klaude, M.; Eriksson, S.; Nygren, J. & Ahnstrom, G.}} (1996): The comet assay: mechanisms and technical considerations. ''Mutation Research'' '''363'''(2): 89-96. <small>{{doi|10.1016/0921-8777(95)00063-1}}</small>
* {{aut|McKelvey-Martin, Valerie J.; Ho, Edwin T.; McKeown, Stephanie R.; Johnston, S. Robin; McCarthy, Patsy J.; Rajab, Nor Fasilah & Downes, C. Stephen}} (1993): Emerging applications of the single cell gel electrophoresis (Comet) assay. I. Management of invasive transitional cell human bladder carcinoma. II. Fluorescent ''in situ'' hybridization Comets for the identification of damaged and repaired DNA sequences in individual cells. ''Mutagenesis'' '''13'''(1): 1-8. [http://mutage.oxfordjournals.org/cgi/reprint/13/1/1 PDF fulltext]
* {{aut|Olive, P.L.; Wlodek, D. & Banath, J.P.}} (1991): DNA double-strand breaks measured in individual cells subjected to gel electrophoresis. ''[[Cancer Research]]'' '''51'''(17): 4671-4676. [http://cancerres.aacrjournals.org/cgi/reprint/51/17/4671.pdf PDF fulltext]
* {{aut|Rojas, E.; Lopez, M.C. & Valverde, M.}} (1999): Single cell gel electrophoresis: methodology and applications. ''Journal of Chromatography B'' '''722'''(1-2): 225-254. <small>{{doi|10.1016/S0378-4347(98)00313-2}}</small> (HTML abstract)
* {{aut|Singh, N.P.; McCoy, M.T.; Tice, R.R. & Schneider, E.L.}} (1988): A simple technique for quantitation of low levels of DNA damage in individual cells. ''Experimental Cell Research'' '''175'''(1): 184-191. <small>{{doi|10.1016/0014-4827(88)90265-0}}</small>
[[Category:Chemical tests]]
[[Category:Electrophoresis]]
[[Category:Molecular biology]]
[[de:Comet Assay]]
[[it:Test della cometa]]