Ethidium bromide intercalation with nucleic acids

The dye 4, 6-Diamidino-2-PhenyIindole (DAPI) in 0.001%W/V aqueous solution can be used directly on smears, ciyosections and embedded specimens to locate and count culture bacteria, without regard to their viability, in cheese and other cultured products. The dye reacts with nucleic acids by intercalation. Excitation at 360nm is best for this dye. It is worth noting two other facts about its use. DAPI cross reacts with dairy proteins, but the color of the protein-dye complex is different from that of the nucleic acid-dye complex (the latter is a steely blue/white) and so the two reactions may be discriminated. The dye also may take up to 15 minutes to enter bacterial cells, particularly spores, before fluorescence is observed. An alternative nucleic acid dye, Ethidium Bromide, has less contrast between the fluorescence induced in cells and the fluorescence of cross-reacting dairy proteins. It should be tried in other products such as meats if DAPI is not successful. 

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,... 

Several complexes that involve intercalation of an acridine in a portion of a nucleic acid have been studied by X-ray crystallographic techniques. These include complexes of dinucleoside phosphates with ethidium bromide, 9-aminoacridine, acridine orange, proflavine and ellipticine (65-69). A representation of the geometry of an intercalated proflavine molecule is illustrated in Figure 6 (b) this is a view of the crystal structure of proflavine intercalated in a dinucleoside phosphate, cytidylyl- -S ) guano-sine (CpG) (70, TV). For comparison an example of the situation before such intercalation is also illustrated in Figure 6 (a) by three adjacent base pairs found in the crystal structure of a polynucleotide (72, 73). In this latter structure the vertical distance (parallel to the helix axis) between the bases is approximately... 

Anyhow, a combination of the Scatchard technique and Raman spectroscopy shows (i) that SOAz actually interacts with DNA at the level of ribose backbones and (ii) that this kind of interaction does not drastically modify the DNA secondary structure, ethidium bromide encountering no more difficulty to intercalate between DNA plates SOAz being grafted or not on the nucleic acid. Thus, the behaviour of MYKO 63 and of SOAz appears quite different with respect to their mode of interaction with DNA despite their close chemical and molecular structure. This surprising observation may be of interest for understanding why SOAz does not induce any cumulative toxicity in vivo in contrast with MYKO 63. 

Intercalators associate with dsDNA by insertion between the stacked base pairs of DNA [52], EtBr binds to dsDNA with little to no sequence specificity, with one dye molecule inserting for every 4-5 base pairs [53]. It also binds weakly via a non-intercalative binding mechanism only after the intercalative sites have been saturated [54], Propidium iodide (PRO) is structurally similar to ethidium bromide, and both dyes show a fluorescence enhancement of approximately 20-30 fold upon binding to dsDNA [41]. As well, their excitation maxima shift 30-40 nm upon binding due to the environment change associated with intercalation into the more rigid and hydrophobic interior of the double-stranded nucleic acid structure relative to aqueous solution [41]. 

Ethidium bromide is a popular stain for nucleic acids, and dye intercalated with DNA during electrophoresis fluoresces when illuminated with UV light. 

Lower nucleic acid concentrations are best determined fluorimetrically. These methods generally depend on the fact that certain dyes can bind to nucleic acids by intercalating between successive base pairs, and this binding is accompanied by marked increases in the fluorescence quantum yield. Ethidium bromide fluorescence (Aex 260-360 nm Af.rr] 560 nm), which is commonly used to visualise nucleic acids in gel electrophoresis, can also be used to quantitate double stranded DNA and RNA with a sensitivity of about 10 ng (Karsten and Wollenberger 1977). The dye 4,6-diamidino-2-phenylindole (DAPI) (Aex 360 nm 2f.m 450 nm) can be used to quantitate DNA specifically with a detection limit of about lng (Brunk et al. 1979). 

Some of these, like ANS and ethidium bromide, will bind non-covalently to particular regions of proteins and nucleic acids, with large changes in their fluorescent properties. ANS lends to bind to hydrophobic patches on proteins and partially unfolded polypeptides, with a blue shift and increase in fluorescence intensity. Ethidium bromide molecules intercalate between the base pairs of double-stranded DNA, resulting in a large increase in fluorescence that is used routinely for detecting and visualizing bands of nucleic acids in gel electrophoresis, for example. 

SYBR dyes for sensitive detection in gels and blots, Chemically reactive SYBR dyes for bioconjugates. The three classes of classic nucleic acid stains are Intercalating dyes (ethidium bromide and propidium iodide), Minor-groove binders, (DAPI and the Hoechst dyes ). Miscellaneous nucleic acid stains with special properties (acridine orange, 7-AAD and hydroxystilbamidine). 39... 

---