Nucleic acid staining dyes are the fluorescent compounds that bind to the nucleic acid; DNA and RNA and help us visualize the nucleic acid in the gel after electrophoresis. Ethidium bromide is one of the oldest known nucleic acid staining dyes available nowadays.
Ethidium bromide
Ethidium bromide (or EtBr) is a fluorescent dye that can be used to stain nucleic acids in molecular biology laboratories. It is an intercalating (getting inserted in between the two strands) agent that intercalates the nucleic acid. Even single-stranded RNA can be detected because it folds back to form local base pairings. The formation of local base pairings in single-stranded RANprovidess sites for the dye to intercalate.
Ethidium bromide can either be incorporated into the agarose gel during the preparation of the gel by adding an appropriate amount of EtBr solution into the gel (precast) or it can be done after running the agarose gel (post electrophoresis staining). After electrophoresis, the gel can also be stained by placing the gel in the EtBr solution followed by destaining it in water. It absorbs the UV irradiation at 210 nm and 285 nm and after excitation, it emits visible light at 605 nm (orange light).
However, it is highly mutagenic and should be handled with extra care. As it is a hydrophobic molecule it can easily pass through the biological membrane (cell membrane and nuclear membrane) and gets inserted into the double-stranded DNA thereby affecting DNA replication and transcription. To substitute EtBr, scientists have developed various other types of dyes that are less toxic but more sensitive than EtBr. These newly developed nucleic acid staining dyes are SYBR® dyes and GelRed/GelGreen dyes.
SYBR® Safe
SYBR® Safe is a member of SYBR® dyes developed by Life Technologies Corporation. It is a cyanine dye that has a structure similar to that of the Ethidium bromide and is 4 to 5 times less toxic than EtBr. The nucleic acid detection sensitivity of SYBR® Safe stain is comparable to that of the EtBr. It has been classified as a non-toxic, non-hazardous waste under U.S. Federal regulation.
Just like the Ethidium bromide, it can be used to stain the agarose gel either during the casting of the gel or after running the electrophoresis. It absorbs the light at 280 nm and 502 nm and emits the light at 530 nm (green light). However, it can enter the cell rapidly which can cause cytotoxicity, therefore it has to be used with appropriate dilution. It is safe to handle and can be disposed of without the need for a complex disposal procedure.
SYBR® Green I and II
These are the cyanine type of nucleic acid staining. Nowadays, researchers use these dyes in place of EtBr. They are much safe in handling and disposing of as compared to that of EtBr. They bind to nucleic acids such as DNA, ssDNA, and RNA; however, RNA staining has a lower performance than that of DNA. SYBR® Green I is for DNA and ssDNA while SYBR® Green II is used for RNA. The DNA-dye binding complex absorbs the blue light at 497 nm and emits the green light at 520 nm.
SYBR ® Gold
SYBR ® Gold is SYBR based nucleic acid staining dye that can be used to stain ssDNA, dsDNA, and RNA. It is the most sensitive fluorescent staining dye available for the detection of all kinds of nucleic acids. SYBR ® Gold is more than 10 times more sensitive than EtBr for detecting nucleic acids in denaturing urea, glyoxal, and formaldehyde gels. It is even more sensitive than SYBR® Green II stain, and silver staining; however, it is less stable than Ethidium bromide.
EvaGreen®
Biotium Inc. Hayward, CA, USA has developed a new nucleic acid staining dye called EvaGreen®. It is a green fluorescent nucleic acid staining dye of superior quality and has high thermal and hydrolytic stability. Because of its stability and lowest inhibitory property towards PCR, it is an excellent replacement for Ethidium bromide or SYBR® Green.
Spectrally, EvaGreen® is much similar to SYBR® Green I. It is usable for many useful applications such as real-time monitoring of qPCR, DNA melt curve analysis, capillary gel electrophoresis, and thermophilic helicase-dependent amplification (tHDA). It is also an intercalating agent and the DNA-dye complex absorbs the light at 500 nm wavelength and emits at 530 nm. You need to incorporate 1 µl of the EvaGreen® dye per 100 µl of the DNA sample prior to load into the wells of agarose gel. It removes the necessity of staining the gel after electrophoresis or incorporating the dye in the gel prior to cast it.
| Name of the dyes | Sample | Absorption peak | Emission peak | Developed by |
|---|---|---|---|---|
| Ethidium Bromide | DNA/RNA | 210 nm (uv) & 285 nm (uv) | 605 nm (orange) | |
| SYBR Safe | DNA/RNA | 280 nm (uv) & 502 nm (green) | 530 nm (green) | Life Technologies Corporation |
| SYBR Green I | DNA | 497 nm (blue) | 520 nm (green) | Life Technologies Corporation |
| SYBR Green II | RNA | 254 nm (uv) & 497 nm (blue) | 520 nm (green) | Life Technologies Corporation |
| SYBR Gold | DNA/RNA | 263 nm (uv) & 496 nm (blue) | 539 nm (green) | Life Technologies Corporation |
| GelRed | DNA/RNA | 280 nm (uv) | 590 nm (orange) | Biotium Inc. |
| GelGreen | DNA/RNA | 254 nm (uv) & 505 nm (green) | 530 nm (green) | Biotium Inc. |
| EvaGreen | DNA | 500 nm (green) | 530 nm (green) | Biotium Inc. |
GelRedTM and GelGreenTM
GelRedTM and GelGreenTM are new-generation fluorescent nucleic acid staining dyes developed by scientists at Biotium Inc. These dyes are superior to EtBr having low toxicity and exceptionally high sensitivity and high thermal and hydrolytic stabilities compared to that SYBR® dyes. These are incapable of entering the living cells as they are unable to pass through the cell membrane unlike the Ethidium bromide and SYBR® dyes. They are noncytotoxic, nonmutagenic, and nonhazardous at a concentration much higher than the working concentration. These stains are suitable for both; precast and post-electrophoresis gel staining.
GelGreenTM is a green fluorescent dye like SYBR® Green and EvaGreen®. It is more sensitive than SYBR® Safe and is environmentally safe. It is suitable for the staining of all types of nucleic acids; ssDNA, dsDNA, and RNA. EvaGreen® absorbs the light at 254 nm and 505 nm wavelengths and emits the green visible light of wavelength 530 nm. GelRedTM is an orange fluorescent dye that absorbs UV light of wavelength 280 nm and emits orange light of wavelength 590 nm. It has similar properties compared to that the GelGreenTM.
