Method for distinguishing normal and cancer cells

• US 5,264,343 A
• Filed: 08/31/1990
• Issued: 11/23/1993
• Est. Priority Date: 08/31/1990
• Status: Expired due to Fees

• Current Assignee: Research Corporation Technologies
• Original Assignee: Eleanor Roosevelt Institute
• Inventors: Krystosek, Alphonse, Puck, Theodore T.
• Primary Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US07/575,900
• Time in Patent Office: 1,180 Days
• Field of Search: 435/6, 435/15, 435/18, 435/91, 435/810, 536/27, 536/28, 536/23.1, 935/77, 935/78
• US Class Current: 435/6.16
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: G01N 33/574 for cancer

Abstract

A method of electing the presence or absence of exposed nuclear DNA is described. Cells are reacted with a reaction composition comprising DNA polymerase I, DNase I, and the nucleotides dATP, dCTP, dGTP, and dTTP or dUTP, at least one of said nucleotides being biotin labeled. Biotin labeled nucleotides incorporated in exposed DNA are detected. Also described is a kit useful for detecting the presence or absence of exposed DNA in cells.

Claims

1. A method to distinguish between normal and transformed or malignant cells by determining the presence or absence of exposed nuclear DNA in mammalian cells, said method comprising the following steps:

(a) reacting mammalian cells with a reaction mixture comprising DNA polymerase I, DNase I, and at least one member selected from the group consisting of the nucleotides dATP, dCTP, dGTP, dTTP and dUTP, at least one of said nucleotides being labeled, to produce reacted cells;

(b) detecting the presence or absence of labeled nucleotides incorporated in exposed DNA in said reacted cells; and

(c) diagnosing for the presence or absence of transformed or malignant cells in said reacted cells, wherein said diagnosing comprises examining the extent and pattern of labelled DNA and comparing said reacted cells with normal cells and cancer cells.

2. The method according to claim 1, wherein said label is biotin or digoxigenin.

3. The method according to claim 2 wherein the biotin labeled nucleotide is biotin-11-dUTP.

4. The method according to claim 2 wherein the biotin labeled nucleotide is biotin-16-dUTP.

5. The method according to claim 19 wherein detecting the presence of biotin labeled nucleotides incorporated in exposed DNA comprises the following steps:

(a) reacting said cells with antibiotin antibodies;

(b) reacting said cells with alkaline phosphatase conjugated antibodies directed against antibiotin antibodies; and

(c) reacting said cells with amplification reagents to obtain a color reaction with alkaline phosphatase conjugated antibodies directed against antibiotin antibodies which are specifically bound to incorporated biotinylated nucleotides.

6. The method according to claim 5 wherein said amplification reagents comprise nitroblue tetrazolium and 5-bromo-4-chloro-3 indolyl phosphate.

7. The method according to claim 19 wherein detecting the presence of biotin labeled nucleotides incorporated in exposed DNA comprises the following steps:

(a) reacting said cells with streptavidin-alkaline phosphatase conjugate; and

(b) reacting said cells with amplification reagents to obtain a color reacting with streptavidin-alkaline phosphatase conjugates which are specifically bound to incorporated biotinylated nucleotides.

8. The method according to claim 7 wherein said amplification reagents comprise nitroblue tetrazolium and 5-bromo-4-chloro-3 indolyl phosphate.

9. The method according to claim 2, wherein said digoxigenin is digoxigenin-11-dUTP.

10. The method according to claim 2 wherein detecting the presence of digoxigenin labeled nucleotides incorporated in exposed DNA comprises the following steps:

(a) reacting said cells with antidigoxigenin antibodies;

(b) reacting said cells with alkaline phosphatase conjugated antibodies directed against antidigoxigenin antibodies; and

(c) reacting said cells with amplification reagents to obtain a color reaction with alkaline phosphatase conjugated antibodies directed against antigigoxigenin antibodies which are specifically bound to digoxigenin labeled nucleotides.

11. The method according to claim 1, wherein said cells are obtained from a fresh biopsy without subsequent in vitro culturing.

12. The method according to claim 1 further comprising the following steps prior to step (a):

(a) precoating coverslips with poly-D-lysine;

(b) seeding said cells onto said coverslips;

(c) placing said coverslips in F12 medium containing 7% fetal calf serum;

(d) rinsing said coverslips twice in phosphate buffered saline;

(e) fixing said coverslips in cold 20;

1 methanol;

acetic acid;

(f) rinsing said coverslips in PBS.

13. A kit useful for distinguishing between normal and transformed or malignant cells by the method of claim 1, which kit comprises the following:

(a) DNA polymerase I, DNase I, and at least one member selected from the group consisting of the nucleotides dATP, dCTP, dGTP, dTTP and dUPT, at least one of said nucleotides being labeled;

(b) a detecting means for detecting the presence of labeled nucleotides incorporated in exposed nuclear DNA; and

(c) a slide of fixed normal cells and a slide of fixed transformed or malignant cells.

14. A kit according to claim 13 wherein said detection means comprises antibiotin antibodies, alkaline phosphatase conjugated antibodies directed against antibiotin antibodies, and amplification reagents.

15. A kit according to claim 12 wherein said detection means comprises streptavidin-alkaline phosphatase conjugate and amplification reagents.

16. A kit according to claims 13 or 14 wherein said amplification reagents comprise nitroblue tetrazolium and 5-bromo-4-chloro-3 indolyl phosphate.

17. The kit according to claim 13, wherein said label is biotin or digoxigenin.

18. A kit according to claim 17 wherein said detection means comprises antigigoxigenin antibodies, alkaline phosphatase conjugated antibodies directed against antidigoxigenin antibodies, and amplification reagents. PG,42

19. A kit for distinguishing between normal and transformed or malignant cells by the method of claim 1, which kit comprises DNA polymerase I, DNase I, and at least one member selected from the group consisting of the nucleotides dATP, dCTP, dGTP, dTTP and dUPT, at least one of said nucleotides being labeled.

20. The kit according to claim 19, wherein said label is biotin or digoxigenin.