DAPI (4',6-diamidino-2-phenylindole) is a DNA-specific probe which forms a fluorescent complex by attaching in the minor grove of A-T rich sequences of DNA. It also forms nonfluorescent intercalative complexes with double-stranded nucleic acids. The physicochemical properties of the dye and its complexes with nucleic acids and history of the development of this dye as a biological stain are described. The application of DAPI as a DNA-specific probe for flow cytometry, chromosome staining, DNA visualization and quantitation in histochemistry and biochemistry is reviewed. The mechanisms of DAPI-nucleic acid complex formation including minor groove binding, intercalation and condensation are discussed.

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DAPI: a DNA-Specific Fluorescent Probe

A method for tracing biochemically defined pathways in the central nervous system using combined fluorescence retrograde transport and immunohistochemical techniques.

In 1885 Carl Rabl published his theory on the internal structure of the interphase nucleus We have tested two predictions of this theory in fibroblasts grown in vitro from a female Chinese hamster, namely (1) the Rabl-orientation of interphase chromosomes and (2) the stability of the chromosome arrangement established in telophase throughout the subsequent interphase Tests were carried out by premature chromosome condensation (PCC) and laser-UV-microirradiation of the interphase nucleus Rabl-orientation of chromosomes was observed in G1 PCCs and G2 PCCs The cell nucleus was microirradiated in G1 at one or two sites and pulse-labelled with 3H-thymidine for 2h Cells were processed for autoradiography either immediately thereafter or after an additional growth period of 10 to 60h Autoradiographs show unscheduled DNA synthesis (UDS) in the microirradiated nuclear part(s) The distribution of labelled chromatin was evaluated in autoradiographs from 1035 cells after microirradiation of a single nuclear site and from 253 cells after microirradiation of two sites After 30 to 60h postincubation the labelled regions still appeared coherent although the average size of the labelled nuclear area fr increased from 142% (0h) to 265% (60h) The relative distance dr, ie the distance between two microirradiated sites divided by the diameter of the whole nucleus, showed a slight decrease with increasing incubation time Nine metaphase figures were evaluated for UDS-label after microirradiation of the nuclear edge in G1 An average of 43 chromosomes per cell were labelled Several chromosomes showed joint labelling of both distal chromosome arms including the telomeres, while the centromeric region was free from label This label pattern is interpreted as the result of a V-shaped orientation of these particular chromosomes in the interphase nucleus with their telomeric regions close to each other at the nuclear edge Our data support the tested predictions of the Rabl-model Small time-dependent changes of the nuclear space occupied by single chromosomes and of their relative positions in the interphase nucleus seem possible, while the territorial organization of interphase chromosomes and their arrangement in general is maintained during interphase The present limitations of the methods used for this study are discussed

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Rabl's Model of the Interphase Chromosome Arrangement Tested in Chinese Hamster Cells by Premature Chromosome Condensation and Laser-UV-Microbeam Experiments

Modification of DAPI banding on human chromosomes by prestaining with a DNA-binding oligopeptide antibiotic, distamycin A.

The properties of two DNA-specific fluorochromes, 4'-6-diamidino-2-phenylindole (DAPI) and mithramycin, have been analyzed as reagents to quantitate cellular DNA by fluorescence microspectrophotometry. Optimal staining conditions and concentrations, and the effects of other cellular materials to which the dyes bind, have been evaluated in measurements of the DNA of rat, chick, and yeast nuclei, Gonyostomum chloroplasts, and T4 particles. Use of either fluorochrome permits a high degree of resolution of different DNA quantities in nuclei and in cell organelles, and the DAPI-DNA complex is sufficiently fluorescent to permit quantitation of the DNA content in genomes as small as those of individual T4 bacteriophage particles. Fluorescence of mithramycin- or DAPI-stained DNA is proportional to DNA quantity when DNA of the same has composition is compared. Quantitation does not appear to be affected discernably by the state of the DNA, whether in different stages of the cell cycle, in condensed chromosomes, or in noncycling, differentiated nuclei. The use of chicken red blood cells is recommended as an internal monitor for variations in staining conditions.

Mithramycin- and 4'-6-diamidino-2-phenylindole (DAPI)-DNA staining for fluorescence microspectrophotometric measurement of DNA in nuclei, plastids, and virus particles.

DIPI and DAPI produce distinct fluorescent bands in human chromosomes similar to quinacrine banding patterns. Additionally, the AT rich secondary constrictions in the chromosomes Nos. 1, 9 and 16 are brightly fluorescent. On the other hand the brilliantly fluorescent regions after staining with quinacrine mustard in the chromosomes Nos. 3 and 4, satellites and some other regions in the acrocentric chromosomes are less striking. The distal part of the Y, however, is clearly discernible. Thus DIPI and DAPI seem to be strictly AT specific fluorochromes like Hoechst 33258.

O. Dann

Papers

DIPI and DAPI: Fluorescence banding with only negligible fading