The Schollar Test

Guest reviewer Dean Madden tests safer alternatives to ethidium bromide for staining DNA on electrophoresis gels.

The stains under test were:


* Methylene blue
* Nile blue sulphate
* Sigma BlueView (TM)
* Azure A
* CarolinaBLU (TM)
* Crystal violet
* Brilliant cresyl blue

   

Ethidium bromide, a potent mutagen
In research laboratories, ethidium bromide and similar fluorescent compounds such as Acridine Orange are normally used to visualise DNA on a gel. Unfortunately, ethidium bromide and its breakdown products are potent mutagens and carcinogens and therefore they should not be used in schools.

Such dyes are often flat molecules with similar dimensions to DNA base pairs. When ethidium bromide binds to DNA, it slips between adjacent base pairs and stretches the double helix. This explains the dye's mutagenic effect - the 'extra bases' cause errors when the DNA replicates. In addition, short-wavelength UV light (which itself is harmful) is required for ethidium bromide to fluoresce and reveal the DNA. For reasons of safety and because UV light of this wavelength causes unwanted mutations in the DNA being studied, several researchers have sought alternative methods of revealing DNA.

Safer alternatives
Crystal violet binds to DNA in a similar way to ethidium bromide and although it is a mutagen, it is not thought to be as harmful as ethidium bromide. Because it can be viewed in normal daylight (avoiding the need for damaging UV light), some researchers have advocated its use where functional DNA is to be recovered from a gel.

Thiazin dyes
The most widely used alternatives to ethidium bromide are methylene blue and its oxidation products, such as Azures A, B and C, Toluidine blue O, Thionin and Brilliant cresyl blue. These dyes are used individually or as mixtures (often in proprietary formulations). Although their exact mode of action is unknown, they are thought to bind ionically to the outside of nucleic acids (to the negatively-charged phosphate groups) and can therefore be used to detect both DNA and single-stranded RNA.

Such dyes are not as sensitive as ethidium bromide, and some of them colour the gel heavily. Consequently, prolonged 'destaining' may be necessary before the DNA bands can easily be seen. Several dyes also fade rapidly after use - methylene blue falls into both categories and is therefore, despite its popularity in school texts, not ideal for staining DNA on a gel.

All of the thiazin dyes may be used in aqueous solution at a concentration of about 0.02-0.04% and applied to the gel after it has been run. They may also be dissolved in mild alkaline solutions (e.g., running buffer; not over about pH 8). Destaining with dilute acetic acid or 0.2 M sodium acetate buffer, pH 4.7 may be necessary for alkaline solutions.

The age of the dye may have a considerable effect upon the results achieved. For example, old samples of methylene blue will almost certainly contain a proportion of other dyes (such as Azures A and B) and these breakdown products may be responsible for much of the staining. Dye solutions are best stored in glass bottles (some dyes will stain plastic containers), either wrapped in foil or kept in the dark.

Staining DNA on the move
Recently, several commercial products have emerged that enable the DNA to be seen as it moves across the gel. Suppliers seldom reveal their composition, but several of these stains contain Nile blue sulphate (also known as Nile blue A), a dye which had not previously been noted for its ability to stain DNA. Adkins and Burmeister (1996) give useful guidance as to its use as well as hints for identifying other dyes which may be useful for visualising DNA. Before it left the schools education market, 
Stratagene used to sell a product called 'Stratabloo', which was amixture of Nile blue sulphate and methylene blue.

All of the dyes used for staining 'mobile' DNA are cationic - that is, they are positively charged in the gel buffer, at pH 8. They move through the gel in the opposite direction to the DNA, latching onto the DNA molecules as they meet them. There exact mode of action is unknown, but, for example, Nile blue sulphate is thought to intercalate within the DNA double helix.

So that sufficient dye remains in the gel, it is added to both the gel and the buffer above it. However, a far lower concentration (1-3 µg per ml) of dye is necessary for this method than for post-electrophoresis staining. This is because too much dye will neutralise the negatively-charged DNA fragments, slowing their movement and reducing the resolution or even preventing the DNA from moving at all. Consequently, there is a compromise to be struck between visibility and resolution. Better results are usually achieved by staining the DNA after the gel has been run, rather than staining during the run.

Drying gels
It is also possible to dry a gel after the dye has been applied, and thereby to concentrate the dye in bands which would otherwise be difficult to see. So that the gel dries evenly, it is advisable to place the wet gel on a sheet of good-quality writing paper, and to place this on several sheets of filter paper. Moisture from the gel soaks into the filter paper, while the writing paper layer stops too much of the dye from soaking out of the gel. Gels should be dried at room temperature.

Safety
Although several dyes that can be viewed in normal daylight are thought to be relatively safe, they have not been as intensively studied as the fluorescent dyes for long-term toxic effects. Some of these visible dyes, apparently, intercalate DNA like ethidium bromide so they too have a potential for mutagenesis and, depending on absorption and metabolism, a potential for carcinogenesis. As with all laboratory chemicals, suitable safety precautions should be exercised when handling any dyes, particularly when they are in dry, powdered form.

   
   
       
   
 

 

Further reading
 
Methylene blue
Yung-Sharp, D. and Kumar, R. (1989) Protocols for the visualisation of DNA in electrophoretic gels by a safe and inexpensive alternative to ethidium bromide. Technique 1 (3) 183-187.
Flores, N. et al (1992) Recovery of DNA from agarose gels stained with methylene blue. Biotechniques 13, 203-205.
 
Brilliant cresyl blue
Santillán Torres, J. and Ponce-Noyoia, P. (1993) A novel stain for DNA in agarose gels Trends in Genetics 9 (2) 40.
 
Nile blue sulphate
Adkins, S. and Burmeister, M. (1996) Visualization of DNA in agarose gels as migrating colored bands: Applications for preparative gels and educational demonstrations Analytical Biochemistry 240 (1) 17-23.
http://www-personal.umich.edu/~steviema/blueDNA.html
 
Crystal violet
Rand, N. (1996) Crystal violet can be used to visualise DNA bands during gel electrophoresis and to improve cloning efficiency Technical Tips Online
http://research.bmn.com/tto
   
   
 

This article is an extended version of one from 'Illuminating DNA' by Dean