Ethidium bromide



Ethidium bromide, (3,8-Diamino-5-ethyl-6-phenylphenanthridine bromide), abbreviated EtBr, is a fluorescent substance used for the detection of nucleic acids in agarose or polyacrylamide electrophoresis. It intercalates between the individual bases of the antiparallel strands of the DNA double helix, but also between the bases of single-stranded DNA and strands of RNA, albeit very weakly, which leads to potential deformation of the strand.

Subsequently, after intercalation between nucleic acid bases and irradiation with UV light fluorescence occurs. Due to its interaction with DNA, EtBr is considered carcinogenic, mutagenic a teratogenic.

Physico-chemical properties
Ethidium bromide is a cationic, aromatic compound with a phenanthridine nucleus. It is the basis of a number of other nucleic acid dyes. The absorption maximum of ethidium bromide depends on the environment in which it occurs - with decreasing polarity of the environment, the fluorescence ability of EtBr increases. In an aqueous environment, it absorbs ultraviolet radiation with a wavelength between 210 and 285 nm and, thanks to its fluorescent properties, then emits orange light with a wavelength of 605 nm. The fluorescence enhancement of EtBr when bound to nucleic acids is attributed to the non-polar nature of the nitrogenous bases themselves, between which it intercalates – due to the repulsion of water molecules, which weaken the fluorescence.

Usage
EtBr was originally used to treat trypanosome infections of cattle. Today's use is mainly in the field of molecular biology to detect double-stranded DNA when evaluating PCR, restriction digestion or the efficiency of DNA isolation. EtBr can be applied directly to the sample, electrophoretic buffer or agarose or polyacrylamide gel. Due to its biological effects, EtBr is used during experiments with neural stem cell transplantation as a gliotoxic agent in animal experiments and focal demyelination.

Health risks
Ethidium bromide is considered a mutagen and carcinogen and it is necessary for laboratories to handle it accordingly and dispose of it properly. Potential risks to laboratory workers when working with EtBr has been under discussion recently however and further research is necessary. Although EtBr intercalates between the DNA double helix, thus deforming it and affecting processes such as replication (inhibition of DNA polymerase and topoisomerase 1) and transcription, the mutagenic nature of the substance itself is not fully proven. In Ames test EtBr is positive, but only after it has been metabolized by liver enzymes from the homogenate. Therefore, it is assumed that the ultimate mutagenic character is due to some EtBr metabolite, which, however, has not yet been proven.