5 Common Dyes for Visualizing and Staining DNA
There are several different stains that can be used to visualize and photograph DNA after the material has been separated by gel electrophoresis.
Among the many choices, these five stains are the most common, beginning with ethidium bromide, which is the most widely used. When working with this process, it's important to not only know the differences between the stains but the inherent health risks.
Ethidium bromide is likely the most well-known dye used for visualizing DNA. It can be used in the gel mixture, the electrophoresis buffer, or to stain the gel after it is run.
Molecules of the dye adhere to DNA strands and fluoresce under UV light, showing you exactly where the bands are within the gel. Despite its advantage, the downside is that ethidium bromide is a potential carcinogen, so it must be handled with great care.
SYBR Gold dye can be used to stain double or single-stranded DNA or to stain RNA. SYBR Gold hit the market as one of the first alternatives to ethidium bromide and is considered to be more sensitive.
The dye exhibits 1000-fold greater UV fluorescence enhancement once it is bound to nucleic acids. It then penetrates thick and high percentage agarose gels and can be used in formaldehyde gels.
Because fluorescence of the unbound molecule is so low, destaining is not required. License-holder Molecular Probes have also (since the launch of SYBR Gold) developed and marketed SYBR Safe and SYBR Green which are safer alternatives to ethidium bromide.
The SYBR Green I and II stains (again, marketed by Molecular Probes) are optimized for different purposes. Because they bind to DNA, they are still considered potential mutagens and because of that, they should be handled with care.
SYBR Green I is more sensitive for use with double-stranded DNA, while SYBR Green II, on the other hand, is best for use with single-stranded DNA or RNA. Like the popular ethidium bromide stain, these highly sensitive stains fluoresce under UV light.
Both SYBR Green I and II are recommended by the manufacturer to be used with "254 nm epi-illumination Polaroid 667 black and white film and a SYBR Green gel stain photographic filter" to achieve detection of 100 pg of RNA or single-strand DNA per band.
SYBR Safe was designed to be a safer alternative to Ethidium Bromide and other SYBR stains. It is not considered hazardous waste and can generally be disposed of through the regular sewer systems (i.e., down the drain), because toxicity testing indicates that there is no acute toxicity.
Testing also indicates there is little or no genotoxicity on Syrian Hamster Embryo (SHE) cells, human lymphocytes, mouse lymphoma cells, or noted in the AMES test. The stain can be used with a blue-light transilluminator which causes less damage to the DNA being visualized and offers better efficiency for later cloning.
Eva Green is a green fluorescent dye that has been found to inhibit Polymerase Chain Reaction (PCR) to a lesser extent than other dyes. This makes it very useful for applications like quantitative real-time PCR.
It is also a good choice if you are using low-melting-point gels for recovery of DNA. It is very stable at high temperatures and has very low fluorescence on its own, but is highly fluorescent when bound to DNA. Eva Green has also been demonstrated to have very low or no cytotoxicity or mutagenicity.