Ethidium Bromide Dyes

Ethidium bromide is an intercalator that is commonly used in molecular biology laboratories as a fluorescent label (nucleic acid stain) for techniques such as agarose gel electrophoresis. It is often abbreviated as EtBr and it is also an abbreviation for Ethyl Bromide. To avoid confusion, some labs use the abbreviation EthBr. When exposed to ultraviolet light, it emits orange fluorescence, which increases the intensity by nearly 20 times when combined with DNA. It has been named ho since the 1950s and is commonly used in veterinary medicine to treat trypanosomiasis in cattle. The high incidence of antimicrobial resistance makes this treatment infeasible in some areas where the relevant isomethylammonium chloride is used. Ethidium bromide may be a mutagen, although it depends on the organisms exposed and the environment.

Ethidium Bromide Dyes Figure 1. Chemical structure of ethidium bromide.

Structure, chemistry, and fluorescence

Like most fluorescent compounds, ethidium bromide is aromatic. Its core heterocyclic part is usually called phenanthridine, and its isomer is the fluorescent dye apyridine. The maximum absorption of EtBr in aqueous solution is at 210 nm and 285 nm, which corresponds to ultraviolet light. As a result of the excitation, EtBr emitted orange light with a wavelength of 605 nm. The strong fluorescence of ethidium bromide after binding to DNA may not be due to the rigid stability of the phenyl moiety, as it has been shown that the benzene ring protrudes outside the inserted base. In fact, the phenyl group was found to be almost perpendicular to the plane of the ring system, as it rotates around its single bond to find a position that will minimally impact the ring system. Instead, the hydrophobic environment found between base pairs is believed to be responsible. By moving into this hydrophobic environment and away from the solvent, ethidium cations are forced to shed all water molecules associated with it. Since water is a highly efficient fluorescence quencher, removing these water molecules can cause ethidium to fluoresce.

Ethidium Bromide Dyes Figure 2. Absorption spectrum of ethidium bromide.

Applications

Ethidium bromide is commonly used in molecular biology laboratories to detect nucleic acids. In the case of DNA, it is usually double-stranded DNA in PCR, restriction digestion, and the like. Single-stranded RNA can also be detected because it usually folds back on itself, providing the insert with local base pairing. Detection usually involves gels containing nucleic acids placed under or under an ultraviolet lamp. Because ultraviolet rays are harmful to the eyes and skin, usually closed gel cameras are used to indirectly observe gels stained with ethidium bromide and record fluorescent images as photographs. Where direct viewing is required, the eyes and bare skin of the viewer should be protected. In the laboratory, when cultures were exposed to mitotic blockers during harvest, intercalation properties have long been used to minimize chromosomal aggregation. The resulting glass slide product allows higher resolution, and therefore is more confident in determining the structural integrity of the chromosome during microscopic analysis.

Ethidium Bromide Dyes Figure 3. DNA sample separated using gel electrophoresis of nucleic acids and stained with ethidium bromide, which emits orange light after binding to DNA.

Reference:

Stevenson, P.; et al. Comparison of isometamidium chloride and homidium bromide as prophylactic drugs for trypanosomiasis in cattle at Nguruman, Kenya. Acta Tropica. 1995, 59 (2): 257–258.

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