- Ethidium bromide
ImageFile = EtBr2.jpg
IUPACName = 3,8-Diamino-5-ethyl-6-phenylphenanthridinium bromide
OtherNames = 2,7-Diamino-10-ethyl-6-phenylphenanthridinium bromide, 2,7-Diamino-10-ethyl-9-phenylphenanthridinium bromide, 3,8-Diamino-1-ethyl-6-phenylphenantridinium bromide, 5-Ethyl-6-phenyl-phenanthridine-3,8-diamine bromide, Ethidium bromide, Homidium bromide, EtBr
Section1 = Chembox Identifiers
CASNo = 1239-45-8
EINECS = 214-984-6
PubChem = 14710
SMILES = [Br-] .CC [n+] 3c4cc(N)ccc4c1ccc(N)cc1c3c2ccccc2
RTECS = SF7950000
KEGG = C11161
Section2 = Chembox Properties
Formula = C21H20BrN3
MolarMass = 394.294 g/mol
Appearance = Purple-red solid
MeltingPt = 260 - 262 °C
Solubility = ~ 40 g/l
Section3 = Chembox Hazards
FlashPt = > 100 °C
NFPA-H = 3
NFPA-F = 0
NFPA-R = 0
RPhrases = R36/37/38, R46
SPhrases = S22, S24/25, S26, S36/37/39, S45, S53
Ethidium bromide (sometimes abbreviated as "EtBr", the abbreviation also confusingly used for
bromoethane) is an intercalating agent commonly used as a nucleic acidstain in molecular biologylaboratories for techniques such as agarose gel electrophoresis. When exposed to ultraviolet light, it will fluoresce with an orange color, intensifying almost 20-fold after binding to DNA. Under the name Homidium, it has been commonly used since the 1950s in veterinary to treat Trypanosomosisin cattle, a disease caused by trypanosomes. Ethidium bromide may be a strong mutagen. It is also widely assumed to be a carcinogenor teratogenalthough this has never been carefully tested.
tructure, chemistry, fluorescence
As with most
fluorescentcompounds, ethidium bromide is aromatic. Its core heterocyclicmoiety is generically known as a phenanthridine, an isomer of which is the fluorescent dye acridine.
The reason for ethidium bromide's intense fluorescence after binding with DNA is probably not due to rigid stabilization of the
phenyl moiety, 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 abut the ring system minimally. Instead, the hydrophobicenvironment found between the base pairs is believed to be responsible. By moving into this hydrophobicenvironment 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 fluorescent quencher, the removal of these water molecules allows the ethidium to fluoresce.
Ethidium bromide is commonly used to detect
nucleic acidsin the lab. In the case of DNAthis is usually double-stranded DNA from PCRs, restriction digests, etc. Single-stranded RNAcan 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 a UV lamp. Since ultravioletlight is harmful to eyes and skin, gels stained with ethidium bromide are usually viewed indirectly using an enclosed camera, with the fluorescentimages 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 utilized 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.
There are alternatives to ethidium bromide in the lab. [ Dean Madden, [http://www.bioscience-explained.org/EN1.2/schollar.html Safer stains for DNA] . accessed 2007-01-31.] For example, several SYBR-based dyes are used by some researchers. SYBR dyes have been found to be less carcinogenic than EtBr and to give cleaner, higher powered staining. However, they are suspended in DMSO, which can rapidly pass through skin. [Huang Q, Fu WL. Comparative analysis of the DNA staining efficiencies of different fluorescent dyes in preparative agarose gel electrophoresis. Clin Chem Lab Med. 2005;43(8):841-2. PMID 16201894] Despite the safety advantage of using SYBR dyes instead of EtBr for staining purposes, many researchers still prefer EtBr for difficult detections.
Ethidium bromide is a
mutagen, suspected carcinogen and at high concentrations is irritating to the eyes, skin, mucous membranes and upper respiratory tractFact|date=April 2007. The health effectsof ethidium bromide exposure have not been thoroughly investigated. It is suspected to be carcinogenicand teratogenicbecause of its mutagenicity, although there is no direct evidence of either effect. The toxic effects of ethidium bromide may be experienced if swallowed, inhaled or absorbed through the skin. However, ethidium bromide is not easily absorbed through the skin because of positive charge and bulky structure. The evidence concerning hazards has been summarized in a [http://rrresearch.blogspot.com/2006/10/heresy-about-ethidium-bromide.html RRResearch post] .
The National Toxicological Program has published data [http://ntp.niehs.nih.gov/index.cfm?objectid=BDAF3AE4-123F-7908-7BE09D1BEA25B435] (this was current as of 08/08/2007) and an Executive Summary [http://ntp.niehs.nih.gov/index.cfm?objectid=6F5EA06A-F1F6-975E-73079A5FE34F7E88] . Both the data sheet and the executive summary note that ethidium bromide (under the name homodium bromide) has "some reported use as an antiparasitic & antiprotazoic drug in animals; has also been used as a drug or drug precursor." In the section "Evidence for Possible Carcinogenic Activity" of the Executive Summary it is stated that: "several early '70s studies reported that EB demonstrated antitumorigenic effects."In the Report on Carcinogens (11th Edition) [http://ntp.niehs.nih.gov/ntp/roc/toc11.html] , ethidium bromide is not listed in the body of the report but is listed in Table 1 with the testing status noted as "No additional testing".
Ethidium bromide is thought to act as a
mutagenbecause it intercalates into double stranded DNA, thereby deforming the molecule. This is believed to block or trip biological processes occurring on DNA, like DNA replicationand transcription.
Preparation of stock solutions and any operations capable of generating ethidium bromide dust or aerosols should be conducted in a
fume hoodto prevent inhalation.Nitrile gloves should be worn at all times. Latex gloves offer little protection from ethidium bromide – gloves made of nitrile rubberare much more effective (this from the manufacturers of nitrile rubber gloves). [ [http://www.safety.ed.ac.uk/resources/General/EthBrHandProt.shtm University of Edinburgh Health & Safety Department - Ethidium Bromide Hand Protection ] ] [ [http://www.purdue.edu/REM/hmm/ethidbr.htm Ethidium Bromide ] ] When working with high concentrations (stocks or powder) or for a prolonged period of time, double gloving can further reduce the risk of exposure, especially if the outer glove is replaced whenever significantly contaminated.
Spills or personal exposure
As with most any chemical, if spilled on skin or eyes, rinse for 15 minutes using a safety shower or eyewash. Gloves should be worn at all times and changed if contaminated with the chemical. If inhaled or swallowed, seek medical attention immediately.
For spills, use a spill pillow or absorbent to soak up aqueous ethidium bromide. Carefully clean up solid ethidium bromide to avoid creating dusts. Place in a sealable container and dispose in a medical waste box.
Ethidium bromide should be handled and disposed of as hazardous waste. This applies to gloves, pipette tips, test tubes, paper towels, etc., that are grossly contaminated with ethidium bromide as well. All ethidium bromide waste should be considered state regulated hazardous waste.
Agarose gel electrophoresis
* Borst P. Ethidium DNA agarose gel electrophoresis: how it started. IUBMB Life. 2005 Nov;57(11):745-7. PMID 16511967
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