Ethidium
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Ethidium bromide (or homidium bromide, chloride salt homidium chloride) is an intercalating agent commonly used as a fluorescent tag ( nucleic acid stain) in
molecular biology Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and phys ...
laboratories for techniques such as
agarose gel electrophoresis Agarose gel electrophoresis is a method of gel electrophoresis used in biochemistry, molecular biology, genetics, and clinical chemistry to separate a mixed population of macromolecules such as DNA or proteins in a matrix of agarose, one of ...
. It is commonly abbreviated as EtBr, which is also an abbreviation for bromoethane. To avoid confusion, some laboratories have used the abbreviation EthBr for this salt. When exposed to ultraviolet light, it will fluoresce with an orange colour, intensifying almost 20-fold after binding to DNA. Under the name homidium, it has been commonly used since the 1950s in veterinary medicine to treat trypanosomiasis in cattle. The high incidence of antimicrobial resistance makes this treatment impractical in some areas, where the related isometamidium chloride is used instead. Despite its reputation as a mutagen, it is relatively safe to handle.


Structure, chemistry, and fluorescence

As with most fluorescent compounds, ethidium bromide is aromatic. Its core heterocyclic moiety is generically known as a phenanthridine, an isomer of which is the fluorescent dye acridine. Absorption maxima of EtBr in aqueous solution are at 210 nm and 285 nm, which correspond to ultraviolet light. As a result of this
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, EtBr emits orange light with wavelength 605 nm. Ethidium bromide's intense fluorescence after binding with DNA is probably not due to rigid stabilization of the phenyl
moiety Moiety may refer to: Chemistry * Moiety (chemistry), a part or functional group of a molecule ** Moiety conservation, conservation of a subgroup in a chemical species Anthropology * Moiety (kinship), either of two groups into which a society is ...
, because the phenyl ring has been shown to project outside the intercalated bases. In fact, the phenyl group is found to be almost perpendicular to the plane of the ring system, as it rotates about its single bond to find a position where it will impinge upon the ring system minimally. Instead, the hydrophobic environment found between the base pairs is believed to be responsible. By moving into this hydrophobic environment and away from the solvent, the ethidium cation is forced to shed any water molecules that were associated with it. As water is a highly efficient fluorescence
quencher In materials science, quenching is the rapid cooling of a workpiece in water, oil, polymer, air, or other fluids to obtain certain material properties. A type of heat treating, quenching prevents undesired low-temperature processes, such as phas ...
, the removal of these water molecules allows the ethidium to fluoresce.


Applications

Ethidium bromide is commonly used to detect nucleic acids in molecular biology laboratories. In the case of DNA this is usually double-stranded DNA from
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s, restriction digests, etc. Single-stranded
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can also be detected, since it usually folds back onto itself and thus provides local base pairing for the dye to intercalate. Detection typically involves a gel containing nucleic acids placed on or under an ultraviolet lamp. Since
ultraviolet Ultraviolet (UV) is a form of electromagnetic radiation with wavelength from 10 nm (with a corresponding frequency around 30  PHz) to 400 nm (750  THz), shorter than that of visible light, but longer than X-rays. UV radiati ...
light is harmful to eyes and skin, gels stained with ethidium bromide are usually viewed indirectly using an enclosed camera, with the fluorescent images recorded as photographs. Where direct viewing is needed, the viewer's eyes and exposed skin should be protected. In the laboratory the intercalating properties have long been used to minimize chromosomal condensation when a culture is exposed to mitotic arresting agents during harvest. The resulting slide preparations permit a higher degree of resolution, and thus more confidence in determining structural integrity of chromosomes upon microscopic analysis. Ethidium bromide is also used during DNA fragment separation by
agarose gel electrophoresis Agarose gel electrophoresis is a method of gel electrophoresis used in biochemistry, molecular biology, genetics, and clinical chemistry to separate a mixed population of macromolecules such as DNA or proteins in a matrix of agarose, one of ...
. It is added to running buffer and binds by intercalating between DNA base pairs. When the agarose gel is illuminated using UV light, DNA bands become visible. Intercalation of EtBr can alter properties of the DNA molecule, such as charge, weight, conformation, and flexibility. Since the mobilities of DNA molecules through the agarose gel are measured relative to a molecular weight standard, the effects of EtBr can be critical to determining the sizes of molecules. Ethidium bromide has also been used extensively to reduce mitochondrial DNA copy number in proliferating cells. The effect of EtBr on mitochondrial DNA is used in veterinary medicine to treat trypanosomiasis in cattle, as EtBr binds molecules of kinetoplastid DNA and changes their conformation to the Z-DNA form. This form inhibits replication of kinetoplastid DNA, which is lethal for trypanosomes. The chloride salt homidium chloride has the same applications. Ethidium bromide can be added to YPD media and used as an inhibitor for cell growth. The binding affinity of the cationic nanoparticles with DNA could be evaluated by competitive binding with ethidium bromide.


Alternatives for gel

There are alternatives to ethidium bromide which are advertised as being less dangerous and having better performance. For example, several SYBR-based dyes are used by some researchers and there are other emerging stains such as "Novel Juice". SYBR dyes are less mutagenic than EtBr by the Ames test with liver extract. However, SYBR Green I was actually found to be more mutagenic than EtBr to the bacterial cells exposed to UV (which is used to visualize either dye). This may be the case for other "safer" dyes, but while mutagenic and toxicity details are available these have not been published in peer-reviewed journals. The MSDS for SYBR Safe reports an for rats of over 5 g/kg, which is higher than that of EtBr (1.5 g/kg). Many alternative dyes are suspended in DMSO, which has health implications of its own, including increased skin absorption of organic compounds. Despite the performance advantage of using SYBR dyes instead of EtBr for staining purposes, many researchers still prefer EtBr since it is considerably less expensive.


Possible carcinogenic activity

Most use of ethidium bromide in the laboratory (0.25–1 µg/ml) is naturally below the LD50 dosage. Testing in humans and longer studies in any mammalian system would be required to fully understand the long-term potential risk ethidium bromide poses to lab workers, but it is clear that ethidium bromide thoroughly mutates and kills both mammalian and bacterial cells.


Handling and disposal

Ethidium bromide is not regulated as hazardous waste at low concentrations, but is treated as hazardous waste by many organizations. Material should be handled according to the manufacturer's Safety Data Sheet (SDS). The disposal of laboratory ethidium bromide remains a controversial subject. Ethidium bromide can be degraded chemically, or collected and incinerated. It is common for ethidium bromide waste below a mandated concentration to be disposed of normally (such as pouring it down a drain). A common practice is to treat ethidium bromide with sodium hypochlorite (bleach) before disposal. According to Lunn and Sansone, chemical degradation using bleach yields compounds which are mutagenic by the Ames test. Data are lacking on the mutagenic effects of degradation products. Lunn and Sansone describe more effective methods for degradation. Elsewhere, ethidium bromide removal from solutions with activated charcoal or ion exchange resin is recommended. Various commercial products are available for this use.


Drug resistance

Trypanosomes in the Gibe River Valley in southwest
Ethiopia Ethiopia, , om, Itiyoophiyaa, so, Itoobiya, ti, ኢትዮጵያ, Ítiyop'iya, aa, Itiyoppiya officially the Federal Democratic Republic of Ethiopia, is a landlocked country in the Horn of Africa. It shares borders with Eritrea to the Er ...
showed universal resistance between July 1989 and February 1993. This likely indicates a permanent loss of function in this area against the tested target, '' T. congolense'' isolated from Boran cattle.


See also

* Phenanthridine *
Agarose gel electrophoresis Agarose gel electrophoresis is a method of gel electrophoresis used in biochemistry, molecular biology, genetics, and clinical chemistry to separate a mixed population of macromolecules such as DNA or proteins in a matrix of agarose, one of ...
and gel electrophoresis of nucleic acids *
GelRed GelRed is an intercalating nucleic acid stain used in molecular genetics for agarose gel DNA electrophoresis. GelRed structurally consists of two ethidium subunits that are bridged by a linear oxygenated spacer. GelRed is a fluorophore, and ...
(itself derived from ethbr) and
GelGreen GelGreen is an intercalating nucleic acid stain used in molecular genetics for agarose gel DNA electrophoresis. GelGreen consists of two acridine orange subunits that are bridged by a linear oxygenated spacer. Its fluorophore, and therefore ...
, marketed as safer and more intense DNA stains *
Propidium iodide Propidium iodide (or PI) is a fluorescent intercalating agent that can be used to stain cells and nucleic acids. PI binds to DNA by intercalating between the bases with little or no sequence preference. When in an aqueous solution, PI has a flu ...
and
propidium monoazide Propidium monoazide (PMA) is a photoreactive DNA-binding dye that preferentially binds to dsDNA. It is used to detect viable microorganisms by qPCR. Visible light (high power halogen lamps or specific LED devices) induces a photoreaction of the ...
, related dyes


References


External links

* {{Commons category-inline Aromatic amines Bromides DNA intercalaters Mutagens Quaternary ammonium compounds Phenanthridine dyes Staining dyes Embryotoxicants Experimental cancer drugs