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Saturday, May 3, 2014

Mechanism of Interaction of Ethidium Bromide with DNA

Ethidium Bromide (EtBr) is an intercalating agent widely used in molecular biology labs as a fluorescent dye for nucleic acid gel electrophoresis .Ethidium Bromide is very sensitive in detecting DNA bands on gel.

etbr structure
Structure of Ethidium Bromide (EtBr)

Mechanism of Interaction of Ethidium Bromide with DNA

Ethidium Bromide contains tricyclic phenanthridine ring system that is able to interact with stacked base pairs of double stranded DNA. Ethidium is capable of forming close van der Walls contacts with the base pairs and due to that it can bind to the hydrophobic interior of the DNA molecule. The peripheral phenyl and ethyl groups projects into the major groove of DNA helix.

intercalation of EtBr with DNA

Approximately one ethidium bromide molecule will get intercalated per 2.5 base pairs, there will be a 20 fold increase in the fluorescence when intercalated. Absorption maxima of EtBr is in the UV range, upon excitation EtBr emits orange light with a wavelength of 605 nm.

EtBr Stained DNA gel / Agarose gel
EtBr Stained Gel

Ethidium Bromide can bind to helical regions formed by  intrastrand base pairing in RNA and in heat denatured or single stranded DNA. The fluorescent yield of DNA-Ethidium Bromide dye complex is many fold higher than the free dye, so it is advantageous in visualizing the DNA on gels. small amounts of DNA ~10ng can be detected using EtBr staining. Ethidium Bromide stock solutions are prepared at a concentration of 0.25  - 1.0 microgram /mL for gel staining.

Ethidium Bromide is considered as a potent mutagen,  it is called as intercalating agent since it can intercalate into the DNA and can interfere with DNA replication and Transcription.

Safer Alternatives
There is still controversies regarding the toxicity of Ethidium Bromide. Even then some researchers prefers to use Ethidium Bromide for nucleic acid staining. But there are some safer alternatives to Ethidium Bromide, here are the commercially available safer dyes: 
  • SYBR Safe, 
  • Gel Red