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| Names | |
|---|---|
| IUPAC name 2-(4-Amidinophenyl)-1H-indole-6-carboxamidine | |
| Other names 4′,6-Diamidino-2-phenylindole | |
| Identifiers | |
3D model (JSmol) | |
| ChEBI | |
| ChEMBL | |
| ChemSpider |
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| UNII | |
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| Properties | |
| C16H15N5 | |
| Molar mass | 277.331 g·mol−1 |
Except where otherwise noted, data are given for materials in theirstandard state (at 25 °C [77 °F], 100 kPa). | |
DAPI (pronounced 'DAPPY', /ˈdæpiː/), or4′,6-diamidino-2-phenylindole, is afluorescentstain that binds strongly toadenine–thymine-rich regions inDNA. It is used extensively influorescence microscopy. As DAPI can pass through an intactcell membrane, it can be used to stain both live andfixed cells, though it passes through the membrane less efficiently in live cells and therefore provides a marker for membrane viability.
DAPI was first synthesised in 1971 in the laboratory of Otto Dann as part of a search for drugs to treattrypanosomiasis. Although it was unsuccessful as a drug, further investigation indicated it bound strongly to DNA and became more fluorescent when bound. This led to its use in identifyingmitochondrial DNA inultracentrifugation in 1975, the first recorded use of DAPI as a fluorescent DNA stain.[1]
Strong fluorescence when bound to DNA led to the rapid adoption of DAPI for fluorescent staining of DNA forfluorescence microscopy. Its use for detecting DNA inplant,metazoa andbacteria cells andvirus particles was demonstrated in the late 1970s, and quantitative staining of DNA inside cells was demonstrated in 1977. Use of DAPI as a DNA stain forflow cytometry was also demonstrated around this time.[1]
When bound to double-stranded DNA, DAPI has an absorption maximum at a wavelength of 358 nm (ultraviolet) and its emission maximum is at 461 nm (blue). Therefore, for fluorescence microscopy, DAPI is excited with ultraviolet light and is detected through a blue/cyan filter. The emission peak is fairly broad.[2] DAPI will also bind toRNA, though it is not as strongly fluorescent. Its emission shifts to around 500 nm when bound to RNA.[3][4]
DAPI's blue emission is convenient for microscopists who wish to use multiple fluorescent stains in a single sample. There is some fluorescence overlap between DAPI and green-fluorescent molecules likefluorescein andgreen fluorescent protein (GFP) but the effect of this is small.
Outside of analytical fluorescence light microscopy DAPI is also popular for labeling ofcell cultures to detect the DNA of contaminatingMycoplasma orvirus. The labelledMycoplasma or virus particles in thegrowth medium fluoresce once stained by DAPI making them easy to detect.[5]
This DNA fluorescent probe has been effectively modeled[6] using thetime-dependent density functional theory, coupled with the IEF version of thepolarizable continuum model. This quantum-mechanical modeling has rationalized the absorption and fluorescence behavior given by minor groove binding andintercalation in the DNA pocket, in term of a reduced structural flexibility and polarization.
DAPI can be used for fixed cell staining. The concentration of DAPI needed for live cell staining is generally very high; it is rarely used for live cells.[7] It is labeled non-toxic in its MSDS[8] and though it was not shown to have mutagenicity toE. coli,[9] it is labelled as a known mutagen in manufacturer information.[2] As it is a small DNA binding compound, it is likely to have somecarcinogenic effects and care should be taken in its handling and disposal.
TheHoechst stains are similar to DAPI in that they are also blue-fluorescent DNA stains which are compatible with both live- and fixed-cell applications, as well as visible using the same equipment filter settings as for DAPI.
