4 ,6 Diamidino-2-phenylindole

R. E. Hicks, R. I. Amann, and D. A. Stahl, Dual staining of natural bacterioplankton with 4, 6-diamidino-2-phenylindole and fluorescent oligonucleotide probes targeting kingdom-level 16S rRNA sequences, Appl. Environ. Microbiol. 571 2158 (1992). 

Fig. 6.8. Structures of some common fluorophores. DPH = diphenylhexatriene NBD = nitrobenzoxadiazole Tb-DTPA-csl24 = terbium diethylenetriaminepentacetate-carbostyril 124, DAPI = 4, 6-diamidino-2-phenylindole Nile red = 9-(diethylamino)-5H-benzo-[a]phenoxazin-5-one.

There are other fluorochromes that can be used for DNA analysis. The following three are the most straightforward and reproducible with regard to application mithramycin, 4, 6-diamidino-2-phenylindole hydrochloride (DAPI), and Hoechst 33258. Concentrations must be determined empirically (see Chapter 30). 

Microbiological Studies. Bacterial numbers for the Chesapeake Bay samples and the pure cultures were determined by acridine orange direct counts (AODC) (67). Those for the lakes were determined by epifluorescence (68) using DAPI (4, 6-diamidino-2-phenylindole) with a final stain concentration of 1.0 xg/mL, a process that is described in detail elsewhere (69). 

Matsuzawa, Y. and Yoshikawa, K. (1994) Change of the higher-order structure in a giant DNA induced by 4 ,6-diamidino-2-phenylindole as a minor groove binder and ethidium bromide as an intercalator. Nucleosides Nucleotides, 13, 1415-1423. 

Other commonly used dyes include Hoechst 33258 and DAPI (4/,6-diamidino-2-phenylindole), both of which show selectivity towards poly A-T sequences. This selectivity limits the utility of these dyes as generic transducers in sensor applications. These dyes associate with double-stranded nucleic acids in a groove-binding motif, more specifically, via binding in the minor groove of B-DNA [41]. 

These methods use fluorescent labels, such as propidium iodide, ethidium bromide, or DAPI (4, 6 -diamidino-2-phenylindole), which are incorporated into the DNA, allowing chromatin condensation and nuclear fragmentation to be visualized under a microscope with the appropriate fluorescence filters. To allow fluorochromes to enter the cells and reach the nucleus, the cells need to be prepermeabilized, for example, with 70% ethanol at -20°C. LMW-DNA fragments may be lost by the permeabilization, decreasing the amount of DNA inside the cells. The lower nucleic acid concentration results in a lower fluorescence intensity in apoptotic cells, which can be detected by fluorescence microscopy or flow cytometry (Calle et al., 2001). 

Fig.8.1a-d. Evans blue extravasation 6 h after permanent middle cerebral artery occlusion (MCAO) in Sprague-Dawley rats. Evans blue extravasation is shown in red and cell nuclei in blue labeled by 4 -6-diamidino-2-phenylindole (DAPI), a compound that binds to DNA. (a) A low-magnification view of the entire ischemic area, (b) Evans blue extravasation from a vessel located in the area adjacent to the ischemic area, (c) Electronic magnification of the box in (b) (arrow is pointing at a leakage of Evans blue from the vessel), (d) No extravasation of Evans blue is seen in a vessel from the same section seen in (b) and (c) but located in the corresponding region of the contralateral hemisphere Contra, contralateral. [Reproduced with permission from Yepes et al. (2003)]... 

Figure 14.2 shows fluorescence intensity quenching of 4,6-diamidino-2-phenylindole (DAPI) complexed to DNA in the presence of two concentrations of Acridine Orange. In fact, one can see that while the fluorescence intensity of DAPI decreases, that of Acridine... 

J. P. F. Tijssen, H. W. Beekes and J. Van Steveninck (1982). Localization of polyphosphate in Saccharomyces fragilis, as revealed by 4,6,-diamidino-2-phenylindole fluorescence. Biochem. 

ProLong Gold antifade reagent with 4 ,6-diamidino-2-phenylindole DAPI (Invitrogen, cat P36931)... 

Chloro-7-nitrobenzo[(3t]-2, 1,3-oxadiazole Dansylchloride 4, 6-Diamidino-2-phenylindole (DAPI)... 

Lower nucleic acid concentrations are best determined fluorimetrically. These methods generally depend on the fact that certain dyes can bind to nucleic acids by intercalating between successive base pairs, and this binding is accompanied by marked increases in the fluorescence quantum yield. Ethidium bromide fluorescence (Aex 260-360 nm Af.rr] 560 nm), which is commonly used to visualise nucleic acids in gel electrophoresis, can also be used to quantitate double stranded DNA and RNA with a sensitivity of about 10 ng (Karsten and Wollenberger 1977). The dye 4,6-diamidino-2-phenylindole (DAPI) (Aex 360 nm 2f.m 450 nm) can be used to quantitate DNA specifically with a detection limit of about lng (Brunk et al. 1979). 

An evanescent wave biosensor was devised for determination of analytes capable of intercalation in dsDNA in a FIA system. A polyethylene lensed optical fiber is coated with a thin polymeric layer containing dsDNA which is immobilized there. The fiber is placed in a FLA system immersed in a solution of ethidium bromide (144), which undergoes intercalation in the dsDNA. The fluorescence signal of 144 is thus enhanced about a 1000-fold relative to the evanescent wave fluorescence measurement without the coating and is dependent on the concentration in solution. If an analyte is present in the same solution, it competes with 144 for intercalation in the DNA and causes fluorescence quenching, which can be measured and correlated to the analyte concentration. This method was applied to determination of various analytes, including 4, 6-diamidino-2-phenylindole dihydrochloride (145)247. 

Hiruki, C. and Rocha, A.D.A. (1986). Hystochemical diagnosis of mycoplasma infections in Catharanthus roseus by means of a fluorescent DNA-binding agent, 4 -6 -diamidino-2-phenylindole-2 HC1 (DAPI). Canadian Journal of Plant Pathology, 8 185-188. 

Sensitive fluorometric assays for double-stranded (native) DNA in tissue extracts are based on the noncovalent interaction of intercalating dyes (Fig. 1.7 such as ethi-dium bromide, 4,6 -diamidino-2-phenylindole (DAPI), or 2-(2-[4-hydroxyphenyl]-6-benzimidazolyl)-6- (1-methyl-4-piperazyl)benzimidazol trihydrochloride (Hoechst 33258), with double-stranded DNA in neutral, aqueous solutions. 

Leusch, H.G., Hoffman, M., and Emeis, C.C. 1985. Fluorometric determination of the total DNA content of different yeast species using 4 6-diamidino-2-phenylindole dihydrochloride. Can. J. Microbiol. 31,1164—1166. 

N. Spackova, T. E. Cheatham III, F. Ryjacek, F. Lankas, L. van Meervelt, P. Hobza, and J. Sponer, Molecular dynamics simulations and thermodynamics analysis of DNA-drug complexes. Minor groove binding between 4, 6-diamidino-2-phenylindole and DNA duplexes in solution, J. Am. Chem. Soc. 125, 1759-1769 (2003). 

Cell nuclei are stained with DAPI (4, 6-diamidino-2-phenylindole) (blue, top and bottom), (f) Purmorphamine (2 pM) induces osteogenesis of mouse multipotent... 

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