Friday, July 20, 2007

Ethidium Bromide Binds to DNA

 
Last Monday's Molecule was ethidium, better known by the name of its common salt, ethidium bromide [Monday's Molecule #35]. Ethidium is a large planer molecule that binds tightly to DNA. It is often used in biochemistry laboratories to visualize fragments of DNA that have been separated on gels. The ethidium molecule is fluorescent—when illuminated with ultraviolet light it shines in the visible range. Here's a picture (below right) of DNA fragments that are illuminated by ethidium binding. It's from an old paper of mine (Moran et al. 1979)—these days you usually can't publish simple experiments like this.

Ethidium binds by inserting itself bewteen the stacked bases in double-stranded DNA. Note that the ring structure of ethidium is hydrophobic and resembles the rings of the bases in DNA. Ethidium is capable of forming close van der Walls contacts with the base pairs and that's why it binds to the hydrophobic interior of the DNA molecule.

Molecules that bind in this manner are called intercalating agents because they intercalate into the compact array of stacked bases. In doing so, they distort the double helix and interfere with DNA replication, transcription, DNA repair, and recombination. This is why intercalating agents are often potent mutagens.

The cartoon below shows the distortion of the sugar-phosphate backbone when an intercalating agent bind and it also shows that the DNA is lengthened when intercalating agents bind. This changes the properties of DNA considerably. One of the tricks in separating closed circular molecules of DNA from linear fragments (such as genomic DNA) is to treat the DNA with ethidium bromide. The intercalating agent doesn't bind to closed circular molecules because they can't be lengthened enough to allow insertion of the chemical between the bases. The normal circular plasmid DNA can then be separated from linear DNA with bound ethidium because binding of ethidium changes the overall density of DNA.



The structure shown above (right) is from Reha et al. (2002). It shows a molecule of ethidium lying between two A/T base pairs.




Moran,L., Mirault, M-E., Tissières, A., Lis, J., Schedl, P., Artavanis-Tsakonas, S., and Gehring, W. (1979) Physical Map of Two D. melanogaster DNA Segments Containing Sequences Coding for the 70,000 Dalton Heat Shock Protein. Cell 17:1-8.

Reha, D., Kabelác, M., Ryjácek, F., Sponer, J., Sponer, J.E., Elstner, M., Suhai, S., and Hobza, P. (2003) Intercalators. 1. Nature of stacking interactions between intercalators (ethidium, daunomycin, ellipticine, and 4',6-diaminide-2-phenylindole) and DNA base pairs. Ab initio quantum chemical, density functional theory, and empirical potential study. J. Am. Chem. Soc. 124:3366-76.

13 comments :

  1. It's worth noting that bound ethidium bromide fluoresces about 25 fold more that unbound EtBr. The fluorescence enhancement arises from increased planarity of the ring system and contributes significantly to detection sensitivity.

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  2. What? Ethidium bromide doesn't bind to circular DNA? When did that happen? Whenever I run plasmids on a gel I can always see the supercoiled and nicked plasmids...

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  3. I guess if EB intercalates in closed circular DNA it induces unwinding, i.e afterwards the DNA is not closed anymore. Thus, the statement that EB doesn't bind to closed circular DNA is correct. See Radloff, Bauer and Vinograd (1967): http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=224502&blobtype=pdf
    Luckily, we do not need to do CsCl density centrifugation anymore.

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  4. EB DOES bind to closed circular DNA, but because the ends are constrained it cannot bind to the extent that it does on linear DNA. This is because it reduces Twist of DNA,which in a plasmid causes increase in writhe (supercoils), which adds torsional stress on the helix and limits the amount of EB that can be bound. Because EB reduces the density of DNA, and plasmids bind less per base-pair than chromosomal (linear) DNA, on a Cesium chloride density gradient the plasmid DNA is slightly more dense than linear DNA,providing a way to purify plasmids. Both plasmid and linear non-constrained DNAs fluoresce red with 300 nm light due to EB bound and can thus easily be identified.

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  5. Thanks for the info. I always enjoy your columns/entries. I am a biochemistry student, struggling to understand the chemistry of life.

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  6. SIR I JUST READ A MECHANISM IN WHICH ETHIDIUM IS USED TO SEPERATE CCC DNA FROM LINEAR DNA,AND I AM AGREE WITH YOUR DISCRIPTION BUT I DIN UNDERSTAND ONE THING GIVEN IN MY BOOK "THE DENSITY OF DNA-ETBR COMPLEX DECREASES AS MORE ETBR BOUND TO IT" COULD U PLEASE EXPLAIN IT?

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  7. As it is already mentioned, EtBr causes unwinding of DNA by intercalating between the base pairs. So, this would obviously cause decrease in the density of the genomic DNA-EtBr complex.

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  8. when we gel extract DNA (after PCR or Restriction digestion) what happens to the EtBr? is it still there bound to the DNA and does it interfere with the other processes we carry out after that

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  9. sir my query is How EtBr bind to the ssDNA. In case of dsDNA it is in the form of intercalaing but how will be the interaction in case of ssDNA.

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  10. Sir, I have also the same question.How ethedium bromide can bind with Single stranded DNA or RNA

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    Replies
    1. Single-stranded oligonucleotides can still have secondary structure when they fold back upon themselves and base pair. EtBr can intercalate between base pairs, whether they are from the same strand or a second strand.

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  11. Could anyone tell me? If the equal concentration of DNA & RNA run in the Gel, Which one will show more visualization & why?

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  12. I have a question Regarding EtBr. If there are two DNA molecules with same size,one is linear and another one is linear with replication bubble. As i Know more EtBr molecules bind to linear DNA strand than molecule with bubble. So, my question is how EtBr distinguishes the shape of the DNA molecule.

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