Fluorescence ethidium bromide

Figure 7.72 Structure of red-orange fluorescing ethidium bromide.

Intercalating dyes 9-aminoacridine (non-fluorescent), ethidium bromide, TOTO, YOYO, YO-PRO-1, TO-PRO-1, TO-PRO-3, SYBR Green I, EnhanCE ... 

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 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. 

The permeability coefficient of 2.6x 10 locm/s at 296 K measured by Deamer is sufficient to supply the enzyme in the liposomes with ADP. How could it be shown that RNA formation actually does take place in the vesicles The increase in the RNA synthesis was detected by observing the fluorescence inside the vesicles. In the interior of the liposomes, the reaction rate is only about 20% of that found for the free enzyme, which shows that the liposome envelope does limit the efficiency of the process. The fluorescence measurements were carried out with the help of ethidium bromide, a fluorescence dye often used in nucleic acid chemistry. 

DNA molecules on gel are stained with ethidium bromide and observed using epi-fluorescent microscopy. 

Ethidium bromide (1) is a widely used guest molecule (Scheme 1). The property that makes 1 a good probe for DNA binding is that its fluorescence quantum yield is very low in water and increases significantly when 1 intercalates between the base pairs of DNA.139... 

Fluorescent stains such as DAPI and ethidium bromide bind to DNA (sometimes even mitochondrial DNA) and have many uses, including location of nuclei and rapid detection of microorganisms or virus formation (22). The lipophilic stain DiOC(6) can be used to stain mitochondria and endoplasmic reticulum in living cells (23,24), and is also a potential-sensitive stain. 

Sivolob, A., De Lucia, F., Revet, B., and Prunell, A. (1999) Nucleosome dynamics II. High flexibility of nucleosome entering and exiting DNAs to positive crossing. An ethidium bromide fluorescence study of mononucleosomes on DNA minicircles. J. Mol. Biol. 285, 1081-1099. 

The scatchard technique consists of studying the difficulties encountered by a fluorescent dye, ethidium bromide (Fig. 19), with respect to its intercalation between the base pairs of DNA when the secondary structure of the macromolecule is modified by binding (or intercalation) of a drug. 

The addition of ethidium bromide (EtBr) or propidium iodide (PI) allows the identification of DNA bands immediately after run on a transilluminator. Intercalation of the dyes into the double-stranded DNA yields fluorescence (EtBr excitation wavelength Aex 302 or 366 nm, emission wavelength Aem 590 nm PI Aex 530 nm, Aem 620 nm). 

Recently, microchip electrophoresis was applied to GAG analysis using ethidium bromide as a fluorescent dye. In particular, separation times were reduced to 150 s, while sensitivity remained comparable to that of conventional electrophoretic methods that rely on cellulose acetate membranes [47]. 

Matsuno Y, Kinoshita M, Kakehi (2005) Fast analysis of glycosaminoglycans by microchip electrophoresis with in situ fluorescent detection using ethidium bromide. J Pharm Biomed Anal 37 429-436... 

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