Ethidium

As an alternative to radiation, a stain such as ethidium bromide is used to visualize DNA. The ethidium may be incorporated into the stmcture of DNA either before or after electrophoresis. The gel is then visualized under a fluorescent lamp. 

The mechanism of interaction with DNA is suggested. Ethidium bromide (EB) displacement assay was performed. We determine the binding constant of Tb-E to DNA to be in the order of Ig K = 6.47 0.4. The bathochromic and hypsochromic effects in the absorption spectra of investigated complex were observed and the interaction is assumed to be mainly of the mono-intercalating type. 

Deoxyribonucleic acid (from plasmids). Purified by two buoyant density ultracentrifugations using ethidium bromide-CsCl. The ethidium bromide was extracted with Et20 and the DNA was dialysed against buffered EDTA and lyophilised. [Marmur and Doty J Mol Biol 5 109 1962 Guerry et al. J Bacteriol II6 1064 1973.] See p. 504. 

Aromatic macrocycles, flat hydrophobic molecules composed of fused, heterocyclic rings, such as ethidium bromide, acridine orange, and actinomycin D... 

The most commonly used dye in fluorescence studies on nucleic acids is ethidium bromide. The dye has broad excitation bands centered around 280 and 460 nm and a strong emission around 600 nm. When the dye hinds to DNA by an intercalative mechanism, its emission is greatly enhanced and slightly shifted in wavelength. In the simplest case with ethidium bromide saturating intercalating sites,... 

The sensitivity of the fluorescence methods varies considerably with the instrument used. Advances in modern instrumentation and the power of today s computers allow for a much improved sensitivity. Using commercially available instruments and modern computers equipped with appropriate software, detection limits down to 10 pg of calf thymus DNA can be achieved using ethidium bromide. (We have achieved such levels using several Perkin-Elmer MPF66 Instruments at various locations.)... 

It should be pointed out that when using ethidium bromide the sensitivity of the assays varies depending on the physical state of the nucleic acids (see Table I). Ethidium does not discriminate between RNA and DNA, although dyes are available which bind DNA exclusively, so the relative amounts of each may be determined by taking two sets of measurements. Alternatively, nucleases (DNA-ase or RNA-ase) can be used to exclusively remove one or the other in a mixture. Nucleic acids from different sources (see Table II) also show a variation in sensitivity, and the fluorescence assay lacks the selectivity of the hybridization technique. Nevertheless, for rapid screening or quality-control applications the fluorescence assay is still the method of choice. 

A typical procedure is shown in Figure 2. Other dyes besides ethidium can be used, although ethidium has an advantage in that its excitation emission bands are well removed from any protein absorbances. A standard curve can be constructed for the nucleic acid of concern and the limits of detection established. In Step 3, proteolytic enzymes may be substituted for heparin, or the step may be bypassed in the case of proteins which do not interfere. After measurement of the unknown sample the nucleic acid concentration may be simply calculated or read from the standard curve. 

Cariello, N., Keohavong, P., Sanderson, B., Thilly, W. DNA damage produced by ethidium bromide staining and exposure to ultraviolet light. Nucleic Acids Research, Vol. 16, No.9, (May 1988), pp. 4157-4161, ISSN 0305-1048... 

Fig. 9 Representative absorption spectra of coralyne with increasing concentrations of calf thymus DNA (a) and relative increase of the viscosities of CCS Col El DNA (b) by coralyne (A) and ethidium ( ). Reprinted from [183] with permission from the publisher...

C19H2 IN7012P-- C21H20M3+-13.5 H20 Ethidium 5-iodouridylyl-(3 — 5 )-adenosine, hydrate ETHIUA10 38 501... 

CioH INdOi jP CaiHaoNa"1" 13.5 HgO 2 Ethidium 5-iodocytidylyl-(3 — 5 )-guanosine, hydrate, ICYGET10 38 502... 

DNA has also been tested as a carrier for ethidium bromide, a drug used in the treatment of protozoal diseases. When compared with free drug, the DNA-bound drug showed decreased toxicity and higher therapeutic efficacy in mice infected with Trypanosoma cruzi [231]. 

Fig. 5 Charge transfer occurs through the DNA /r-stack and is strongly dependent on minor base stack perturbations. Photoinduced electron transfer is observed from tethered intercalated ethidium to a rhodium intercalator bound to DNA up to 30 A away. The efficiency of electron transfer is drastically reduced in the presence of a stacking disruption, here a single base-pair mismatch... 

Fig. 6 Dynamic molecular motions can gate DNA-mediated charge transport. Two time constants (5 and 75 ps) are observed for hole transfer from photoexcited ethidium, tethered and intercalated near the end of a 14-base pair DNA duplex, to a base analog, 7-deazaguanine, in DNA. The 5 ps time constant, which is independent of distance between 10-17 A, is due to direct hole transfer, while the 75 ps time constant corresponds to reorientation of the ethidium before hole transfer. Adapted from [96]... 

In DNA RNA hybrids, where intrastrand base stacking is comparable to B-DNA, we observed efficient long range guanine oxidation when using ethidium as an intercalating photooxidant [130]. Conversely, a Rh(III) com-... 

Oxidative repair is not a unique feature of our Rh(III) complexes. We also demonstrated efficient long-range repair using a covalently tethered naphthalene diimide intercalator (li /0 1.9 V vs NHE) [151]. An intercalated ethidium derivative was ineffective at dimer repair, consistent with the fact that the reduction potential of Et is significantly below the potential of the dimer. Thymine dimer repair by a series of anthraquinone derivatives was also evaluated [151]. Despite the fact that the excited triplets are of sufficient potential to oxidize the thymine dimer ( 3 -/0 1.9 V vs NHE), the anthraquinone derivatives were unable to effect repair [152]. We attribute the lack of repair by these anthraquinone derivatives to their particularly short-lived singlet states anthraquinone derivatives that do not rapidly interconvert to the excited triplet state are indeed effective at thymine dimer repair [151]. These observations suggest that interaction of the dimer with the singlet state may be essential for repair. 

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