AAT Bioquest

What are the most commonly used intercalating DNA dyes?

Posted January 6, 2022


Most commonly used intercalating DNA dyes include:

Ethidium Bromide – This intercalating agent experiences a roughly 20-fold increase in brightness on binding to DNA and is excellent for staining DNA in agarose gel electrophoresis. It fluoresces under UV light. 

SYBR Green – This highly sensitive DNA stain fluoresces under ultraviolet light. Helixyte™ Green has the same spectral properties to those of SYBR® Green. Helixyte™ Green has much greater sensitivity for dsDNA, thus especially useful for assays where the presence of contaminating RNA or ssDNA might obscure results.

SYBR Safe – The major advantages of this DNA dye are that it is as sensitive as ethidium bromide and can be visualized without UV light. A blue light box offers a safer option for visualization as the wavelengths do not cause DNA damage. SYBR® Safe was introduced as a safer alternative to EtBr and SYBR® Green, but unfortunately, it is much less sensitive than SYBR® Green. It only has sensitivity comparable to EtBr. Gelite™ Safe has been developed specifically to be less hazardous than EtBr for staining DNA in agarose and acrylamide gels with much higher sensitivity. Gelite™ Safe has greatly improved safety and uncompromised sensitivity. 

Propidium Iodide – This intercalating DNA dye exhibits a 20-30 fold increase in fluorescence on binding to DNA. The dye is membrane impermeable and can only enter cells with compromised membranes, making it an excellent probe for identifying dead cells.

SYBR Gold – On binding to nucleic acids, this highly sensitive intercalating DNA dye exhibits 1000-fold greater UV fluorescence.

Crystal Violet – This is a highly sensitive dye that is detectable in the visible range, which eliminates the risks of UV exposure. It intercalates with DNA in a similar manner as ethidium bromide but is less mutagenic. It is an excellent option for detection of nucleic acid in gel electrophoresis. 

DAPI (4',6-diamidino-2-phenylindole) – DAPI binds strongly to A-T rich regions of dsDNA. On binding it exhibits a roughly 20-fold increase in fluorescence. Its inability to pass through intact cell membranes easily makes DAPI more successful at staining dead cells or cells with compromised membranes rather than live cell staining.

7-AAD (7-aminoactinomycin D) – 7-AAD binds strongly to the G-C rich regions of double-stranded DNA. Its large stokes shift makes it effective for use in multicolor analysis in conjunction with blue and green fluorescent probes.

Gel Red – A robust, stable and sensitive DNA dye, Gel Red can be used as an in-gel stain or post stain. It is visualized using UV light.

Additional resources

Nanomechanics of Fluorescent DNA Dyes on DNA Investigated by Magnetic Tweezers

Gel Electrophoresis