US4423153A
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US
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Methods and compositions for the detection and determination of cellular DNA
Assignee: The University of Texas System
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Filing: 1981-12-03
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Granted: 1983-12-27
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Expires: 2001-12-03
Abstract
Abstract Fluorochrome compositions and methods for the selective detection and determination of double stranded, helical DNA are provided. DNA is quantitated by determining the fluorescence enhancement imparted from the formation of a highly specific DNA-fluorochrome adduct. The fluorochrome composition disclosed includes a divalent metallic cation and a fluorescent antibiotic, antineoplastic agent selected from mithramycin, chromomycin or olivomycin. A compatible surfactant can also be included with the fluorochrome composition to enhance solubilization of cellular matter. Moreover, a compatible membrane penetrating agent such as dimethyl sulfoxide can be included with the composition to accelerate solubilization of the cellular matter. The compositions and methods provided are also useful for the screening or quantitating of agents which interact with DNA to induce displacement of fluorochrome bound to DNA.
Inventors
- 👤 David F. Ranney
- 👤 Alfred J. Quattrone
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-26 01:03
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/52; Y; Y10; Y10T; Y10T436/00; Y10T436/25
Claims Analysis
Total Claims: 46
Claim 1
Independent Claim
73 words
A fluid composition, adapted for use in selectively detecting and determining the cellular content of double stranded, helical DNA in the presence of cellular debris, comprising in aqueous solution: a divalent metallic cation cofactor; a fluorochrome which in the presence of the cofactor is capable of significant fluorescence enhancement upon interaction with double-stranded, helical DNA; a small concentration of a compatible surfactant which is effective to enhance solubilization of DNA and cellular debris.
Claim 2
Dependent Claim
73 words
A fluid composition, adapted for use in selectively detecting and determining the cellular content of double stranded, helical DNA in the presence of cellular debris, comprising in aqueous solution: a divalent metallic cation cofactor; a fluorochrome which in the presence of the cofactor is capable of significant fluorescence enhancement upon interaction with double-stranded, helical DNA; a small concentration of a compatible surfactant which is effective to enhance solubilization of DNA and cellular debris.
Claim 3
Dependent Claim
32 words
The fluid composition in accordance with claim 1 wherein said divalent metallic cation is selected from the group consisting of Mg +2 , Mn +2 , Zn +2 and Co +2 .
Claim 4
Dependent Claim
17 words
The fluid composition in accordance with claim 1 wherein the divalent metallic cation is Mg +2 .
Claim 5
Dependent Claim
20 words
The fluid composition in accordance with claim 1 wherein the divalent metallic cation is in the form of a salt.
Claim 6
Dependent Claim
18 words
The fluid composition in accordance with claim 1 wherein the fluorochrome is an antibiotic containing a chromomycinone complex.
Claim 7
Dependent Claim
23 words
The fluid composition in accordance with claim 1 wherein the fluorochrome is selected from the group consisting of aureolic acid, chromomycin, and olivomycin.
Claim 8
Dependent Claim
13 words
The fluid composition in accordance with claim 1 wherein the fluorochrome is mithramycin.
Claim 9
Dependent Claim
20 words
The fluid composition in accordance with claim 1 wherein the compatible surfactant is a salt of a higher alkyl sulfonate.
Claim 10
Dependent Claim
16 words
The fluid composition in accordance with claim 8 wherein the higher alkyl sulfonate is dodecyl sulfonate.
Claim 11
Dependent Claim
19 words
The fluid composition in accordance with claim 1 which further comprises a small concentration of a compatible penetrating agent.
Claim 12
Dependent Claim
15 words
The fluid composition in accordance with claim 10 wherein the penetrating agent is dimethyl sulfoxide.
Claim 13
Independent Claim
112 words
A method for selectively detecting and determining the intracellular content of double stranded, helical DNA in the presence of cellular debris comprising: providing a sample cell culture; solubilizing the cell culture; admixing the solubilized cell culture with a fluid composition comprising a divalent metallic cation cofactor, and a fluorochrome which in the presence of the cofactor is capable of significant fluorescence enhancement upon interaction with double stranded, helical DNA; and determining the extent of fluorescence enhancement by comparing the fluorescence of the admixed solubilized cell culture and fluid composition relative to fluorescence of the fluid composition alone, said fluorescence enhancement being a measure of the intracellular content of double stranded helical DNA.
Claim 14
Independent Claim
112 words
A method for selectively detecting and determining the intracellular content of double stranded, helical DNA in the presence of cellular debris comprising: providing a sample cell culture; solubilizing the cell culture; admixing the solubilized cell culture with a fluid composition comprising a divalent metallic cation cofactor, and a fluorochrome which in the presence of the cofactor is capable of significant fluorescence enhancement upon interaction with double stranded, helical DNA; and determining the extent of fluorescence enhancement by comparing the fluorescence of the admixed solubilized cell culture and fluid composition relative to fluorescence of the fluid composition alone, said fluorescence enhancement being a measure of the intracellular content of double stranded helical DNA.
Claim 15
Dependent Claim
25 words
The method in accordance with claim 12 wherein the divalent metallic cation is Mg +2 , Mn +2 , Zn +2 or Co +2 .
Claim 16
Dependent Claim
17 words
The method in accordance with claim 12 wherein the fluorochrome is an antibiotic containing a chromomycinone complex.
Claim 17
Dependent Claim
25 words
The method in accordance with claim 12 wherein the fluorochrome is an antibiotic compound selected from the group consisting of aureolic acid, chromomycin, and olivomycin.
Claim 18
Dependent Claim
12 words
The method in accordance with claim 12 wherein the fluorochrome is mithramycin.
Claim 19
Dependent Claim
25 words
The method in accordance with claim 12 wherein the fluid composition comprises Mg +2 as the divalent metallic cation cofactor and mithramycin as the fluorochrome.
Claim 20
Dependent Claim
15 words
The method in accordance with claim 12 wherein the cell culture is solubilized by sonification.
Claim 21
Dependent Claim
26 words
The method in accordance with claim 12 wherein the fluid composition further includes a compatible surfactant, the surfactant effective to enhance solubilization of the cell culture.
Claim 22
Dependent Claim
15 words
The method in accordance with claim 19 wherein the higher alkyl sulfonate is dodecyl sulfonate.
Claim 23
Dependent Claim
17 words
The method in accordance with claim 12 wherein the fluid composition further includes a compatible penetrating agent.
Claim 24
Dependent Claim
14 words
The method in accordance with claim 21 wherein the penetrating agent is dimethyl sulfoxide.
Claim 25
Independent Claim
145 words
A method for assessing lymphocyte activation, wherein the intracellular content of DNA is a measure of lymphocyte activation, the method comprising: providing a lymphocyte cell culture sample; stimulating the lymphocyte cell culture sample with a mitogen or antigen; incubating the stimulated cell culture for a suitable period of time sufficient to induce intracellular synthesis of DNA; solubilizing the mitogen stimulated lymphocyte cell culture after the incubation period; admixing the solubilized cell culture with a fluid composition comprising a divalent metallic cation cofactor, and a fluorochrome which in the presence of the cofactor is capable of significant fluorescent enhancement upon interaction with double stranded helical DNA; and determining the extent of fluorescence enhancement by comparing the fluorescence of the admixed solubilized cell culture and fluid composition relative to the fluorescence of the fluid composition alone, said fluorescence enhancement being a measure of intracellular double-stranded helical DNA.
Claim 26
Independent Claim
145 words
A method for assessing lymphocyte activation, wherein the intracellular content of DNA is a measure of lymphocyte activation, the method comprising: providing a lymphocyte cell culture sample; stimulating the lymphocyte cell culture sample with a mitogen or antigen; incubating the stimulated cell culture for a suitable period of time sufficient to induce intracellular synthesis of DNA; solubilizing the mitogen stimulated lymphocyte cell culture after the incubation period; admixing the solubilized cell culture with a fluid composition comprising a divalent metallic cation cofactor, and a fluorochrome which in the presence of the cofactor is capable of significant fluorescent enhancement upon interaction with double stranded helical DNA; and determining the extent of fluorescence enhancement by comparing the fluorescence of the admixed solubilized cell culture and fluid composition relative to the fluorescence of the fluid composition alone, said fluorescence enhancement being a measure of intracellular double-stranded helical DNA.
Claim 27
Dependent Claim
25 words
The method in accordance with claim 23 wherein the divalent metallic cation is Mg +2 , Mn +2 , Zn +2 or Co +2 .
Claim 28
Dependent Claim
17 words
The method in accordance with claim 23 wherein the fluorochrome is an antibiotic containing a chromomycinone complex.
Claim 29
Dependent Claim
25 words
The method in accordance with claim 23 wherein the fluorochrome is an antibiotic compound selected from the group consisting of aureolic acid, chromomycin, and olivomycin.
Claim 30
Dependent Claim
12 words
The method in accordance with claim 23 wherein the fluorochrome is mithramycin.
Claim 31
Dependent Claim
25 words
The method in accordance with claim 23 wherein the fluid composition comprises Mg +2 as the divalent metallic cation cofactor and mithramycin as the fluorochrome.
Claim 32
Dependent Claim
15 words
The method in accordance with claim 29 wherein the cell culture is solubilized by sonification.
Claim 33
Dependent Claim
26 words
The method in accordance with claim 23 wherein the fluid composition further includes a compatible surfactant, the surfactant effective to enhance solubilization of the cell culture.
Claim 34
Dependent Claim
15 words
The method in accordance with claim 30 wherein the higher alkyl sulfonate is dodecyl sulfonate.
Claim 35
Dependent Claim
17 words
The method in accordance with claim 23 wherein the fluid composition further includes a compatible penetrating agent.
Claim 36
Dependent Claim
14 words
The method in accordance with claim 32 wherein the penetrating agent is dimethyl sulfoxide.
Claim 37
Independent Claim
141 words
A method for the determination of the extent of binding to a double-stranded helical DNA of a test compound relative to displacement of DNA bound fluorochrome, the method comprising: providing double stranded, helical DNA; providing said test compound; providing a composition comprising a divalent metallic cation cofactor, and the fluorochrome which in the presence of the cofactor is capable of fluorescence enhancement upon binding with double stranded helical DNA; admixing said compound and composition together with double-stranded, helical DNA under conditions suitable for the binding of both the test compound and fluorochrome to double-stranded, helical DNA; and determining the extent of binding of the test compound to double-stranded, helical DNA by comparing the difference of the fluorescence associated with the admixture containing the test compound, composition and DNA relative to the fluorescence associated with a combination of the composition and DNA.
Claim 38
Independent Claim
141 words
A method for the determination of the extent of binding to a double-stranded helical DNA of a test compound relative to displacement of DNA bound fluorochrome, the method comprising: providing double stranded, helical DNA; providing said test compound; providing a composition comprising a divalent metallic cation cofactor, and the fluorochrome which in the presence of the cofactor is capable of fluorescence enhancement upon binding with double stranded helical DNA; admixing said compound and composition together with double-stranded, helical DNA under conditions suitable for the binding of both the test compound and fluorochrome to double-stranded, helical DNA; and determining the extent of binding of the test compound to double-stranded, helical DNA by comparing the difference of the fluorescence associated with the admixture containing the test compound, composition and DNA relative to the fluorescence associated with a combination of the composition and DNA.
Claim 39
Dependent Claim
25 words
The method in accordance with claim 34 wherein the divalent metallic cation is Mg +2 , Mn +2 , Zn +2 or Co +2 .
Claim 40
Dependent Claim
17 words
The method in accordance with claim 34 wherein the fluorochrome is an antibiotic containing a chromomycinone complex.
Claim 41
Dependent Claim
25 words
The method in accordance with claim 34 wherein the fluorochrome is an antibiotic compound selected from the group consisting of aureolic acid, chromomycin, and olivomycin.
Claim 42
Dependent Claim
12 words
The method in accordance with claim 34 wherein the fluorochrome is mithramycin.
Claim 43
Dependent Claim
25 words
The method in accordance with claim 34 wherein the fluid composition comprises Mg +2 as the divalent metallic cation cofactor and mithramycin as the fluorochrome.
Claim 44
Dependent Claim
16 words
The method in accordance with claim 34 wherein the composition is provided as an aqueous solution.
Claim 45
Dependent Claim
21 words
The method in accordance with claim 34 wherein the composition is provided as a dried dispersion on a solid-phase support material.
Claim 46
Dependent Claim
16 words
The method in accordance with claim 41 wherein the solid-phase support material is a cellulosic material.
References Cited (Backward Citations)
US4302437A
Cited
US4345027A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1986-12-26 | 1987-06-27 | 1987-12-27 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1990-12-26 | 1991-06-27 | 1991-12-27 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1994-12-26 | 1995-06-27 | 1995-12-27 | $7700.00 | Window Closed / Lapsed |