Ethidium bromide alternatives

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. 

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. 

Intercalators with asymmetric substituents, such as the phenyl and ethyl groups of ethidium bromide (21), frequently cause a smaller increase in DNA length than expected from the simple model described above. In such cases these groups are inserted into the minor groove of the DNA helix with concomitant bending of the double helix towards the major groove. This alternative type of complexation is supported by X-ray studies on model systems, 25). 

The final concentration of the cRNA transcript can be determined by its absorbance. The absorbance of a 1 1000-2000 dilution of the transcript is read at a wavelength of 260 nm (one unit A260 is equivalent to 40 pg/mL of RNA). Alternatively, densitometry on ethidium bromide-stained agarose gels or colorimetric stains (e.g., RiboGreen, Molecular Probes, Inc., Eugene, OR) can be used to... 

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. 

Starting with the first IPCR study, gel electrophoresis retains its potential as a fast and easy method for end-point determination of DNA amplificate for IPCR assays [10, 24, 25, 29, 31, 35, 36, 38, 39, 64], Readout is performed by intercalation fluorescence markers (e.g., ethidium bromide) and photometric/densitometric quantification of band signal intensities. The direct addition of a double-strand specific intercalation marker to the PCR amplificate and subsequent measurement of fluorescence in microwells proved to be of insufficient sensitivity for the quantification of IPCR amplificate [37]. Alternative approaches, such as radioactive labeling during PCR and subsequent imaging [33], were carried out but are not well suited for routine clinical application because of additional methodological requirements. An advantage of gel electrophoresis is the possibility of simultaneous amplificate detection for multiplex IPCR [41] and the ability to detect nonspecific amplification products. 

After amplification, tlie products can be detected by various methods. Simple gel electrophoresis with ethidium bromide staining may suffice. When greater accuracy is required, one of the primers can be fluorescently labeled so that after PCR the fragments are accurately sized on a DNA sequencing device. Alternatively, some form of hybridization assay can be used to verify or analyze the amplified product. Automated methods are always attractive and closed-tube methods are particularly advantageous in the clinical laboratory. Adding a fluorescent dye or probe before amplification allows thermocyclers equipped with optical detection to analyze the reaction as it progresses (real-time PCR) or after the reaction is complete (endpoint measurement) without need to process the sample for a separate analysis step. 

Bands or spots of radioactive DNA in gels can be visualized by autoradiography. Alternatively, a gel can be stained with ethidium bromide, wliich fluoresces an intense orange when bound to a double-helical DNA molecule (Figure 5-2), A band containing only 50 ng of DNA can he readily seen. 

Figure 5. Yeast Chromosomal Trsmslocation Analysis. Ethidium bromide-stained gel. The first lane was loaded with size markers eus in Figure 4. Other lanes were alternately loaded with wild-type yeast (no translocations) and a strain containing a translocation between chromosomes 3 and 12. Experimental conditions were as in Figure 3, using 1.5% low endoosmosis agarose gel. The arrow indicates the position of the wild-type chromosome and the absence of the translocated chromosome.

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