US5455157A
Expired / Lapsed
US
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Method for the non-radioactive measurement of the nucleic acid synthesis in eukaryotic cells
Assignee: Roche Diagnostics GmbH
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Filing: 1994-04-19
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Granted: 1995-10-03
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Expires: 2014-04-19
Abstract
Abstract For the determination of the rate of nucleic acid synthesis in eukaryotic cells a nucleotide which is non-radioactively labelled or derivatized with a hapten is introduced into the cells with the addition of liposomes and the rate of synthesis of the nucleic acids is determined by means of the incorporation of the nucleotide via its label or derivatization.
Inventors
- 👤 Matthias Hinzpeter
- 👤 Herbert von der Eltz
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 23:37
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; Y; Y10; Y10S; Y10S436/00; Y10S436/829; Y10T; Y10T436/00; Y10T436/14; Y10T436/142222; Y10T436/143333
Claims Analysis
Total Claims: 21
Claim 1
Independent Claim
74 words
Method for the determination of the rate of nucleic acid synthesis in a eukaryotic cell, comprising introducing a nucleotide which is non-radioactively labelled or derivatized with a hapten into said cell with the addition of a cationic liposome which does not encapsulate said nucleotide and determining the rate of nucleic acid synthesis by determining incorporation of the non-radioactively labelled or derivatized nucleotide into nucleic acids of said eukaryotic cell as a function of time.
Claim 2
Dependent Claim
74 words
Method for the determination of the rate of nucleic acid synthesis in a eukaryotic cell, comprising introducing a nucleotide which is non-radioactively labelled or derivatized with a hapten into said cell with the addition of a cationic liposome which does not encapsulate said nucleotide and determining the rate of nucleic acid synthesis by determining incorporation of the non-radioactively labelled or derivatized nucleotide into nucleic acids of said eukaryotic cell as a function of time.
Claim 3
Dependent Claim
63 words
Method as in claim 1, wherein said nucleotide is derivatized with a hapten which is a first member of a binding pair and said method further comprises determining the rate of nucleic acid synthesis by adding a labelled, second member of the binding pair which binds to said first member in said eukaryotic cell and determining binding between said first and second member.
Claim 4
Dependent Claim
18 words
Method as in claim 2, wherein said second member of said binding pair is a labelled anti-hapten antibody.
Claim 5
Dependent Claim
55 words
Method as in claim 1 wherein said nucleotide is a pyrimidine nucleotide which has been labelled or derivatized at the 5-position or a purine nucleotide labelled or derivatized at the 8-position, or a 7-deaza purine nucleotide labelled or derivatized at the 7-position, said nucleotide having as a sugar residue a ribose, deoxyribose or dideoxyribose unit.
Claim 6
Dependent Claim
11 words
Method as in claim 4, wherein said nucleotide is 5-digoxigenin-2-'-deoxyuridine triphosphate.
Claim 7
Dependent Claim
39 words
Method as in claim 2 wherein said second member of said binding pair is labelled with an enzyme and said method further comprises adding a substrate for said enzyme to said eukaryotic cell and determining cleavage of said substrate.
Claim 8
Dependent Claim
14 words
Method as in claim 6, wherein said enzyme is β-galactosidase, alkaline phosphatase or peroxidase.
Claim 9
Dependent Claim
36 words
Method as in claim 1 further comprising culturing the eukaryotic cells in a cell culture medium having serum added to said culture medium with the introduction of the cationic liposomes and said labelled or derivatized nucleotide.
Claim 10
Dependent Claim
21 words
Method as claimed in claim 1, further comprising determining said rate of nucleic acid synthesis by immunocytochemical examination of proliferating cells.
Claim 11
Dependent Claim
20 words
Method as in claim 1, further comprising determining said rate of nucleic acid synthesis via an enzyme linked immunosorbent assay.
Claim 12
Dependent Claim
11 words
Method of claim 1, wherein said cationic liposome comprises N-[1(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride.
Claim 13
Dependent Claim
22 words
Method of claim 2, wherein said second member of said binding pair is labelled with a fluorescent dye or a luminescent dye.
Claim 14
Dependent Claim
14 words
Method of claim 12, wherein said fluorescent dye is resorufin, fluorescein, coumarin or rhodamine.
Claim 15
Dependent Claim
19 words
Method of claim 3, wherein said labelled anti-hapten antibody is labelled with a fluorescent dye or a luminescent dye.
Claim 16
Dependent Claim
25 words
Method of claim 6, wherein said second member of said binding pair is a labelled, anti-hapten antibody and said nucleotide is derivatized by a hapten.
Claim 17
Dependent Claim
78 words
Method for the determination of the rate of nucleic acid synthesis in a eukaryotic cell, comprising introducing a nucleotide which is derivatized with a hapten selected from the group consisting of digoxigenin, biotin and fluorescein into said cell with the addition of a cationic liposome which does not encapsulate said nucleotide and determining the rate of nucleic acid synthesis be determining incorporation of the derivatized nucleotide into nucleic acids of said eukaryotic cell as a function of time.
Claim 18
Dependent Claim
78 words
Method for the determination of the rate of nucleic acid synthesis in a eukaryotic cell, comprising introducing a nucleotide which is derivatized with a hapten selected from the group consisting of digoxigenin, biotin and fluorescein into said cell with the addition of a cationic liposome which does not encapsulate said nucleotide and determining the rate of nucleic acid synthesis be determining incorporation of the derivatized nucleotide into nucleic acids of said eukaryotic cell as a function of time.
Claim 19
Dependent Claim
76 words
Method for the determination of the rate of nucleic acid synthesis in a eukaryotic cell, comprising introducing a nucleotide which has been non-radioactively labelled with a fluorescent dye or a luminescent dye into said cell with the addition of a cationic liposome which does not encapsulate said nucleotide and determining the rate of nucleic acid synthesis by determining incorporation of the non-radioactively labelled nucleotide into nucleic acids of said eukaryotic cell as a function of time.
Claim 20
Dependent Claim
76 words
Method for the determination of the rate of nucleic acid synthesis in a eukaryotic cell, comprising introducing a nucleotide which has been non-radioactively labelled with a fluorescent dye or a luminescent dye into said cell with the addition of a cationic liposome which does not encapsulate said nucleotide and determining the rate of nucleic acid synthesis by determining incorporation of the non-radioactively labelled nucleotide into nucleic acids of said eukaryotic cell as a function of time.
Claim 21
Dependent Claim
24 words
Method as in claim 17 further comprising determining said rate of nucleic acid synthesis by measuring immunofluorescence of proliferating cells in an immunofluorescence assay.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
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| 3.5 Years (Year 4) | 1998-10-02 | 1999-04-03 | 1999-10-03 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2002-10-02 | 2003-04-03 | 2003-10-03 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2006-10-02 | 2007-04-03 | 2007-10-03 | $7700.00 | Window Closed / Lapsed |