JPS6361622B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for detecting nucleic acids in biological samples. The present invention relates generally to fluorescence microscopy, which has recently been developed as an important tool for biological research. Fluorescence microscopy is an extremely sensitive method for the detection of trace amounts of substances that can be stained with dyes that fluoresce when excited by incident light. In particular, the present invention relates to methods for detecting various organisms in biological samples. Detection of the above-mentioned organisms is possible due to the nucleic acid staining property of the fluorescent dye of the present invention. It is clear from the scientific literature that a variety of compounds are used for the selective coloring of nucleic acids. Huon Bertalanhuy and Bitkis are J.Histochen.
Cytochem. vol. 4, 481-493 (1956) reported that acridine orange can be used for the identification of basophilia (RNA) in cell chambers by fluorescence microscopy. In particular, it has been shown that acridine orange allows the identification of chamber inclusions of Basophilia in a supravital state, and that the red-fluorescent chamber inclusions are stained by the toluidine blue method and shown by ribonucleic acid enzymes to be composed primarily of RNA. They have discovered that they can respond to the things that they are exposed to. Lotsell et al. Nature, vol. 253, 461 (1975)
reported that 4'-6-diamidino-2-phenylindole (DAPI) was shown to have useful DNA binding properties. In particular, we have discovered that DAPI can be used as a very specific fluorescent colorant for both nuclear and threadonal DNA in yeast. Hillwig and Grotsup are Experimental Cells.
Research 75, 122-126 (1972) A simple and direct fluorescence method to visualize the location of heterochromatic chromosomal sections and repetitive DNA (in mice) using a benzimidazole derivative (designated Hoechst 33258). Describes the coloring method. Although conventional dyes appear to be widely used in fluorescence microscopy, histology, and microfluorometry,
These have undesirable properties in terms of excitation and emission spectra, quantum yield, and substrate fluorescence, which has prompted the search for alternative fluorescent nucleic acid dyes. There is therefore a clear need for specific dyes for nucleic acids that can be excited at visible wavelengths and are essentially nonfluorescent in their free state, but highly fluorescent when bound to the nucleic acid. . Compounds useful in the present invention are generally described in U.S. Pat.
3833863 and Defense Publication No. T88 9016 as a dye. However, there is no description or proposal regarding new uses of the present invention. Specifically, the present invention relates to a novel method for detecting nucleic acids, which method comprises the formula: (In the formula, R represents an alkyl group having 1 to 4 carbon atoms and the above alkyl group in which the alkyl moiety is substituted with dialkylamino having 1 to 4 carbon atoms, and R ₁ and R ₂ each represents an alkyl group having 1 to 4 carbon atoms,

[Formula] represents a heterocyclic nucleus selected from the group consisting of a benzothiazole nucleus, a quinoline nucleus, a pyridine nucleus, and a substituted pyridine nucleus in which the alkyl moiety is substituted with dialkylamino having 1 to 4 carbon atoms, and X is an anion. It is characterized by staining nucleic acids in biological samples with a fluorescent dye represented by ion (representing an ion). Although the dyes used in the method of the invention are essentially non-fluorescent in aqueous solution, the dyes
shows significant enhancement when combined with Although some of these dyes do not show this enhancement when bound to monofilamental DNA, most dyes clearly show a significant increase in fluorescence when bound to RNA. Since there is no fluorescence in the free state, large amounts of dye can be used without the need to separate or remove excess reagent. This property is important in applications where dye absorption is poor. Furthermore, the high fluorescence quantum yield of some dyes allows the use of smaller amounts of dye if necessary. The following dyes were prepared and tested for use in the present invention:

【table】

[Table] Lysinium iodide

[Table] Dinium iodide was irradiated to measure the fluorescence emission, which can be considered as the excitation wavelength of the above dyes. DNA and RNA samples were then stained with each dye. The sample was irradiated with an appropriate wavelength and fluorescence enhancement was observed. The results are shown in the table.

【table】

[Table] All the above data are Perkin-Elmer
0.1 μM in 3 ml of 10 mmol phosphate buffered saline (PBS) PH7.3 using an MPF43-A fluorescence spectrophotometer.
It is made using dyes. The ratio indicating fluorescence enhancement is the ratio of the fluorescence intensity of a dye solution containing 100 μg per milliliter of calf thorax type DNA and yeast type RNA to a dye solution containing no dye. Relative fluorescence is simply obtained from the ratio of the fluorescence intensity of a dye solution containing 100 μg per milliliter of calf thorax DNA to the intensity of a dye solution containing the same amount of DNA. Data are not corrected for instrument response at different wavelengths. Example 1 5 μ of a dye solution (2 mg/ml in 0.9% salt solution) was added into 100 μ of fresh whole blood (EDTA). The mixture was shaken and wet slides were quickly prepared for examination under a fluorescence microscope (540 nm broad band excitation filter, 590 nm long pass emission filter, 400x magnification). Nuclei of peripheral blood leukocytes (PBLs) were visible and stained intense orange against a dark background. In addition, platelets stained with moderate intensity were observed. No staining of the cell chambers of red blood cells and PBLs was observed. Example 2 Dye solution (2 mg/ml in 0.9% salt solution) 20μ
was added to 2 ml of E. coli suspension (10 ⁸ -10 ⁹ cells/ml). The mixture was stirred and wet slides were prepared and tested as described in Example 1. Bacteria were visible as glowing orange sticks against a black background. Example 3 20 μ of dye No. 25 solution (2 mg/ml) was added to 2 ml of E. coli suspension (~10 ⁹ cells/ml). When the slides were examined as described in Example 1, the bacteria were visible as bright red rods against a black background. Example 4 Phage suspension (PICM, 5×10 ⁹ pfu/ml;
T6, 4× ¹⁰⁸ pfu/ml; 2× ¹⁰¹⁰ pfu/ml) 100μ
dye solution (1.6 mg/ml in 0.9% salt solution, 0.3 μm
10μ of sterile filtered solution was added. The mixture was shaken to prepare wet slides. The slides were examined under a fluorescence microscope as in Example 1. PICM and T6 were seen as dots of orange light against a black background. Example 5 Stock solutions of DNA and dye in 50 μM phosphate PH6.8 buffer to a final concentration of 1-2 ml in a final volume of
Mix to make 100nm DNA base pairs and 1μM dye. Perkin-Elmer Fluorescence in Solutions
Measured on MPF43A. Excitation was at 555 nm (4 nm bandpass). The emission was at 605nm (4nm bandpass). A linear curve of fluorescence versus DNA concentration was obtained over this range. Example 6 To 5μ of fresh whole blood (EDTA) was added 0.5ml of dye No. 28 solution (50μg/ml in 0.9% salt solution, sterile filtered through a 0.3μm filter). Shake the mixture and apply a fluorescence microscope (450-490nm broadband excitation, 530nm long-pass emission filter, magnification
A wet slide was prepared for examination under 400×).
The nuclei of peripheral blood leukocytes are stained bright orange;
The nuclear reticulum was stained yellow, and the plasma and membranes were stained green. The small plates were dyed orange. Red blood cells and reticulocytes can be easily distinguished because the latter's reticulum is strongly stained yellow-orange, and the red blood cell membrane is weakly stained green. Example 7 The method of Example 6 was repeated using Dye No. 33 solution. The nuclei of peripheral blood leukocytes and the nuclear reticulum were strongly stained yellow, and the plasma and membranes were stained intensely green-yellow.
Red blood cells and reticulocytes can be easily distinguished by the intensely stained yellow reticulocyte reticulum and green-yellow stained red blood cell membranes. It is clear from the above examples that the dyes demonstrating the use of the present invention can be used to examine an almost unlimited variety of microscopic organisms in biological samples. Besides their obvious use in microfluorocytology, these dyes can be used in flow cell fluorescence analyzers for testing urine and water samples for high bacterial concentrations. These dyes are used for plasmids, reticulocytes,
Whole blood can also be analyzed to determine component status, as white blood cells and platelets are fluorescently stained.

Claims (1)

[Claims] 1. Nucleic acids with the formula: (In the formula, R represents an alkyl group having 1 to 4 carbon atoms and the above alkyl group in which the alkyl moiety is substituted with dialkylamino having 1 to 4 carbon atoms, and R ₁ and R ₂ each represents an alkyl group having 1 to 4 carbon atoms,
[Formula] represents a heterocyclic nucleus selected from the group consisting of a benzothiazole nucleus, a quinoline nucleus, a pyridine nucleus, and a substituted pyridine nucleus in which the alkyl moiety is substituted with dialkylamino having 1 to 4 carbon atoms, and X is an anion. 1. A method for detecting nucleic acids in a biological sample, which comprises staining with a fluorescent dye (representing an ion). 2. The method according to claim 1, wherein the fluorescent dye is 1-γ-dimethylaminopropyl-4-p-dimethylamino-styrylquinolinium iodide. 3. The method according to claim 1, wherein the fluorescent dye is 3-γ-dimethylaminopropyl-2-p-dimethylamino-styrylbenzothiazolium iodide. 4. The method according to claim 1, wherein the fluorescent dye is 1-γ-dimethylaminopropyl-4-p-dimethylaminostyrylpyridinium iodide. 5. The method according to claim 1, wherein the fluorescent dye is 1-methyl-2-dimethylamino-4-p dimethylaminostyrylpyridinium iodide.