The 10 Best DNA Dyes and Probes

With over 50 different probes on the market, it can be a bit overwhelming to determine the optimal DNA dye for a given experiment. To simplify the process, here is what we consider the 10 best DNA dyes.

Ethidium Bromide

  • cheap
  • potential hazard
  • well-established
  • impermeable
  • gel electrophoresis
  • fluorescence microscopy
  • orange fluorescence

 

Ethidium bromide (EtBr) is one of the most popular DNA dyes. This is because it was one of the first to be commercially available. As early as the 1950s, it was used to treat diseases in livestock. In the 1970s, scientists began using it as a DNA probe. In addition to its early adoption, ethidium bromide is comparatively inexpensive. Even in large quantities, it remains very affordable, running around $30 per gram, but depending on the vendor and the packaging size the prices can be substantially higher or lower. Ethidium bromide is excellent for staining DNA in agarose gel electrophoresis. It can also be used to detect dsDNA in PCRs. Upon binding to DNA, EtBr experiences a roughly 20-fold increase in brightness. It releases an orange fluorescence (605 nm) when excited by UV light (~300 nm). Ethidium bromide can also detect RNA depending on how much RNA folding occurs. Ethidium bromide is not a good probe for detecting DNA in live cells, however. this is because it is impermeable to intact cell membranes. Over the years, health concerns have arisen over ethidium bromide usage. In particular, concerns have been raised about ethidium bromide's role as a mutagen, due to it being an intercalating agent. This has spurred intense debate as to whether ethidium bromide is optimal for conventional laboratory use. But as of now no studies have supported this claim. Ethidium bromide might interfere with DNA replication and transcription in humans when exposed in large quantities. At low concentrations it is not considered a hazardous waste.

Propidium Iodide

  • flow cytometry
  • multiplexing
  • red fluorescence
  • 488 nm argon-ion laser
  • dead cell
  • impermeable

 

Propidium iodide is a DNA dye and an intercalating agent. It is in the same chemical family as ethidium bromide. Like ethidium bromide, propidium iodide has a nitrogen-containing ring structure that forms its core. It differs in that it has an additional quaternary amine which binds ionically to an iodide ion. Upon binding to DNA, propidium iodide will experience a 20-30 fold increase in fluorescence. It can also bind to RNA if folding occurs. Propidium iodide is membrane impermeable. It can only enter cells with compromised membranes. This makes it an excellent probe for identifying dead cells. It can also be used to quantitatively assess DNA content in a biological sample. Propidium iodide has no sequence preference and binds roughly once per 4-5 base pairs. It can be excited by a xenon- or mercury- lamp as well as a 488 nm argon-ion laser. Because of its emission at 617 nm, it can easily be used in multiplex assays. It can be combined with green fluorescent probes such as fluorescein. It can also be used as a counterstain for multicolor analysis. In terms of price, it is significantly more expensive than ethidium bromide (about $1000 per gram). However, it is a very common dye for flow cytometry. It can also be used for fluorescence microscopy and fluorescence spectroscopy.

Crystal Violet

  • gram stain
  • gel electrophoresis
  • non-fluorescent
  • poor sensitivity
  • cheap
  • non-toxic

 

Crystal violet is a simple chemical compound consisting of three bound benzene rings attached to three amines. It has long been in use for various medical purposes, serving as both an antibacterial agent and a topical antiseptic. It also has a long history of use as a histological stain, with early uses dating back to the late 1800's. Crystal violet is perhaps most well known for its use in Gram staining, to determine if bacteria are gram positive or gram negative. As a DNA dye, crystal violet can be used for nucleic acid detection in gel electrophoresis. In these applications, it serves as an alternative to fluorescent staining reagents such as ethidium bromide. This is a particularly powerful advantage as use of ultraviolet light sources to excite fluorescent probes may lead to DNA degradation. The tradeoff for such applications of crystal violet, however, is a loss of sensitivity. In experiments, crystal violet has been shown to be less sensitive to DNA than fluorescent probes such as ethidium bromide. Whereas ethidium bromide can detect as little as 1 ng of DNA in a gel band, crystal violet sensitivity sits at around 16 ng. This sensitivity can be improved to 8 ng if a counterstain, such as methyl orange, is used. However, it will still not be as sensitive as ethidium bromide. In terms of pricing, crystal violet is very affordable. It is also fairly non-toxic. If health concerns are an issue, crystal violet can serve as a non-toxic alternative to some of the more hazardous compounds such as ethidium bromide.

dUTP-conjugated Probes

  • versatile
  • direct labeling
  • hybridization
  • PCR
  • FISH
  • DIG-dUTP

 

dUTP-conjugated probes form an interesting class of DNA detectors mainly resulting from the experimental flexibility they provide. The basic idea is to attach a probe to dUTP with what's called a linker. Then, the dUTP is incorporated into DNA through molecular techniques. This also incorporates the probe into the DNA macromolecule. In this way, the DNA becomes labeled with the probe. Because dUTP can be conjugated with many different probes, dUTP-conjugates have a wide range of potential applications. For example, dUTP probes can be used to monitor PCR reactions. They can also be used as the probe in FISH procedures. In general, dUTP probes excel in situations where DNA hybridization takes place. Some of the most common dUTP conjugates include labeling with compounds like digoxigenin (DIG), biotin and fluorescein. Labeling with these compounds all provide non-radioactive probes for DNA detection. These probes in particular have also found widespread use in immunoassays like ELISA.

DAPI (4',6-diamidino-2-phenylindole)

  • blue fluorescence
  • A-T sequence preference
  • fluorescence microscopy
  • slightly permeable
  • cytotoxic
  • non-toxic
  • multiplex

 

DAPI, like many of the dyes on this list, was first synthesized as a medical agent. It was initially used in an attempt to treat trypanosomiasis, a disease caused by parasites. In the late 1970s, DAPI was adopted for use as a DNA probe due to its efficacy in binding DNA and its large subsequent increase in fluorescence. DAPI binds particularly strongly to A-T rich regions of double-stranded DNA and will experience a roughly 20-fold increase in fluorescence upon binding. It can also bind to RNA, however the fluorescence increase is weaker and the emission is red-shifted. DAPI is commonly used for fluorescence microscopy. It is particularly successful at staining dead cells or cells with compromised membranes. DAPI can pass through intact cell membranes, albeit with difficulty. Thus, it is not recommended for live cell staining. DAPI has an excitation of 358 nm and an emission of 461 nm. This means it will appear blue when visualized with a blue/cyan filter. It can also be used in conjunction with green-fluorescent probes such as GFP. In experiments, DAPI has high cytotoxicity, which reinforces the reason to avoid using DAPI for live cell staining. DAPI is fairly non-toxic to humans if exposure occurs. However, as with many of the DNA probes, it is possible for DAPI to have some mutagenic properties.

7-AAD (7-aminoactinomycin D)

  • G-C sequence preference
  • multiplexing
  • red fluorescence
  • 543 nm helium-neon laser
  • dead cells
  • impermeable
  • fluorescence microscopy
  • flow cytometry

 

7-AAD is a fluorescent probe and intercalating agent. Like DAPI, it has a strong affinity for binding double stranded DNA. Unlike DAPI which binds to A-T rich regions, 7-AAD selectively binds to G-C rich regions. It will also bind to RNA, so digestion enzymes may be required before staining. 7-AAD has an excitation of 546 nm and an emission of 647 nm. Because of its large Stokes shift, many researchers use 7-AAD for multicolor analysis in conjunction with blue and green fluorescent probes. 7-AAD is well excited by a 543 nm helium-neon laser. 7-AAD is suitable for use in detecting dead cell populations or cells with compromised membranes. It does not readily pass through intact membranes, making it a poor stain for live cells. 7-AAD has found large use in fluorescence microscopy and flow cytometry.

Hoechst 33258 (33342, 34580)

  • blue fluorescence
  • live cell
  • membrane permeable
  • low cytotoxicity
  • A-T sequence preference
  • xenon-mercury lamps
  • UV laser
  • fluorescence microscopy
  • flow cytometry

 

The Hoechst stains are a group of blue-fluorescent DNA probes. They have been used to stain DNA as early as the 1970s. Developed by the German company Hoechst AG, these dyes offer a good alternative to traditional blue fluorescent dyes. The Hoechst stains offer significantly greater cell permeability than analogous dyes such as DAPI. This means that Hoechst stains, such as Hoechst 33258, are suitable for staining both live and dead cells. Furthermore, because of the Hoechst dyes' lower cytotoxicity, the impact to live cell populations is greatly reduced. Hoechst stains will selectively bind to A-T rich regions of double-stranded DNA, specifically binding to the minor groove. Upon binding to DNA, Hoechst stains will undergo an approximately 30-fold increase in fluorescence. The Hoechst stains can be excited by ultraviolet light (~360 nm) and will emit a blue fluorescence (~460 nm). These dyes are compatible with xenon-mercury lamps as well as UV lasers. They are excellent for applications in fluorescence microscopy, immunohistochemistry and flow cytometry. The difference between Hoechst 33258, 33342 and 34580 are important. Compared to Hoechst 33258, Hoechst 33342 is significantly more permeable due to the addition of a lipophilic ethyl group. For Hoechst 34580, the emission maximum is slightly blue-shifted, residing at 437 nm compared to the 461 nm emission maximum of Hoechst 33258 and Hoechst 33342.

YOYO-1/DiYO-1/TOTO-1/DiTO-1

  • extremely sensitive
  • high cost
  • impermeable
  • flow cytometry
  • cell viability
  • cyanine
  • green fluorescence

 

These are a family of cyanine dyes based around the compound oxazole yellow. Despite the name however, these dyes actually fluoresce green rather than yellow. For example, YOYO-1 (and its chemical equivalent, DiYO-1) has a maximum emission of 509 nm. The same is true of TOTO-1 (and its chemical equivalent, DiTO-1); this compound has a maximum emission of 535 nm. These compounds are intercalating agents, meaning they will insert themselves between the planar surfaces of DNA base pairs. This family of DNA probes is particularly well known for its high affinity for DNA. Upon binding to DNA, these probes can experience a one thousand to three thousand fold increase in fluorescence. This strong affinity for DNA makes them excellent for experiments which require great sensitivity. The trade-off though is that these probes are fairly expensive. One mL may cost upwards of a few thousand dollars. Because these probes are cell-impermeable, they cannot pass through intact cell membranes. They are well suited for staining fixed or dead cells. In this regard, these probes have been commonly used in cell viability and cytotoxicity assays in conjunction with platforms such as flow cytometry.

Dyes and Stains at Biomol