Method for the non-radioactive measurement of the nucleic acid synthesis in eukaryotic cells

• US 5,455,157 A
• Filed: 04/19/1994
• Issued: 10/03/1995
• Est. Priority Date: 05/22/1989
• Status: Expired due to Fees

• Current Assignee: Boehringer Mannheim GmbH (Roche Holding AG)
• Original Assignee: Boehringer Mannheim GmbH (Roche Holding AG)
• Inventors: von der Eltz, Herbert, Hinzpeter, Matthias
• Primary Examiner(s): SCHWARTZ, RICHARD A
• Assistant Examiner(s): Lin, Ching-I Patsy
• Application Number:
  US08/231,364
• Time in Patent Office: 532 Days
• Field of Search: 435/6, 435/7.1, 436/63, 436/94, 436/501, 436/829, 935/76, 935/77, 935/78, 264/4.1, 264/4.3, 536/23.1
• US Class Current: 435/6.18
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: Y10S 436/829 Liposomes, e.g. encapsulation
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

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.

Claims

1. 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.

2. 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.

3. Method as in claim 2, wherein said second member of said binding pair is a labelled anti-hapten antibody.

4. 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.

5. Method as in claim 4, wherein said nucleotide is 5-digoxigenin-2-'"'"'-deoxyuridine triphosphate.

6. 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.

7. Method as in claim 6, wherein said enzyme is β

-galactosidase, alkaline phosphatase or peroxidase.

8. 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.

9. Method as claimed in claim 1, further comprising determining said rate of nucleic acid synthesis by immunocytochemical examination of proliferating cells.

10. Method as in claim 1, further comprising determining said rate of nucleic acid synthesis via an enzyme linked immunosorbent assay.

11. Method of claim 1, wherein said cationic liposome comprises N-[1(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride.

12. Method of claim 2, wherein said second member of said binding pair is labelled with a fluorescent dye or a luminescent dye.

13. Method of claim 12, wherein said fluorescent dye is resorufin, fluorescein, coumarin or rhodamine.

14. Method of claim 3, wherein said labelled anti-hapten antibody is labelled with a fluorescent dye or a luminescent dye.

15. 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.

16. 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.

17. 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.

18. Method as in claim 17 further comprising determining said rate of nucleic acid synthesis by measuring immunofluorescence of proliferating cells in an immunofluorescence assay.