Physical separation of nucleic acids by antibodies to halogenated nucleotides

• US 6,417,343 B1
• Filed: 10/16/1997
• Issued: 07/09/2002
• Est. Priority Date: 10/17/1996
• Status: Expired due to Fees

• Current Assignee: New York Medical College (Touro College & University System)
• Original Assignee: New York Medical College (Touro College & University System)
• Inventors: Darzynkiewicz, Zbigniew, Juan, Gloria, Traganos, Frank
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US08/951,598
• Time in Patent Office: 1,727 Days
• Field of Search: 435/6, 435/7.1, 435/91.1, 530/388.21, 530/391.1, 530/391.3, 935/77, 536/25.3, 536/25.4
• US Class Current: 536/25.3
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites

Abstract

Methods for immunocytochemical detection and isolation of RNA.

Claims

1. A method for labeling RNA by incorporating a 5-position halogenated pyrimidine into the RNA by the steps of:

(i) incubating the RNA with 5-position halogenated pyrimidine under conditions whereby the incorporation can occur, (ii) incubating the RNA having incorporated therein a portion of said 5-position halogenated pyrimidine, the incorporated form being a monophosphate pyrimidine, as formed in step (i) with anti-halogenated pyrimidine antibody which binds specifically to said halogenated pyrimidine providing for a means for labeling RNA (iii) separating the labeled RNA from non-labeled RNA.

2. The method of claim 1 wherein said 5-position halogenated pyrimidine is 5-position halogenated uridine triphosphate.

3. The method of claim 1 wherein said 5-position pyrimidine is 5-position brominated uridine.

4. The method of claim 1 wherein said antihalogenated pyrimidine is a labeled antibody.

5. The method of claim 4 wherein the label in said labeled antibody is selected from the group consisting of a fluorochrome label, a peroxidase label, a digoxigen label and a biotin label.

6. The method of claim 1 wherein said antibody is a monoclonal antibody.

7. A method for labeling RNA by incorporating a 5-position halogenated pyrimidine into the RNA by the steps of:

(i) incubating the RNA with 5-position halogenated pyrimidine under conditions whereby the incorporation can occur, (ii) incubating the RNA having incorporated therein a portion of said 5-position halogenated pyrimidine, the incorporated form being a monophosphate pyrimidine, as formed in step (i) with anti-halogenated pyrimidine antibody which binds specifically to said halogenated pyrimidine providing for a means of separating such RNA wherein said antibody is bound to a solid support.

8. The method of claim 7 wherein said solid support is a member selected from the group consisting of particles, plates, microspheres, beads, magnetized microspheres and fibers.

9. A method for labeling RNA by incorporating a 5-position halogenated pyrimidine into the RNA by the steps of:

(i) incubating the RNA with 5-position halogenated pyrimidine under conditions whereby the incorporation can occur, (ii) incubating the RNA having incorporated therein a portion of said 5-position halogenated pyrimidine, the incorporated form being a monophosphate pyrimidine, as formed in step (i) with anti-halogenated pyrimidine antibody which binds specifically to said halogenated pyrimidine providing for a means of separating such RNA further comprising adding at least one enzyme to step (i) which acts to facilitate the incorporation of said 5-position halogenated pyrimidine into the RNA.

10. A method for isolating RNA which comprises the steps of, (i) incubating the RNA with a 5-position halogenated pyrimidine under conditions whereby the incorporation can occur, (ii) incubating the RNA having incorporated therein a portion of said 5-position halogenated pyrimidine, the incorporated portion being a monophosphate, as formed in step (i) with an antihalogenated pyrimidine antibody which binds specifically to said halogenated pyrimidine and which is attached to a solid support, (iii) isolating said solid support and (iv) separating the RNA from said antibody to isolate the RNA.

11. The method of claim 10 wherein said 5-position halogenated pyrimidine is 5-position halogenated uridine triphosphate.

12. The method of claim 11 wherein said 5-position halogenated pyrimidine is 5-position brominated uridine.

13. The method of claim 11 wherein said antihalogenated pyrimidine is a labeled antibody.

14. The method of claim 13 wherein the label in said labeled antibody is selected from the group consisting of a fluorochrome label, a peroxidase label, a digoxigen label and a biotin label.

15. A method for separating RNA from DNA which comprises the steps of separately incorporating a 5-position halogenated pyrimidine and 5-position halogenated deoxy pyrimidine into the RNA and DNA respectively, by the step of (i) incubating the RNA and DNA with a 5-position halogenated pyrimidine and a 5-position halogenated deoxy pyrimidine under conditions whereby the incorporation of the 5-position halogenated pyrimidine into the RNA and the incorporation of the 5-position halogenated deoxy pyrimidine into the DNA can occur, (ii) incubating the RNA and DNA each having incorporated therein a portion of said 5-position halogenated pyrimidine and said 5-position halogenated deoxy pyrimidine, respectively, the incorporated portion being a monophosphate as formed in step (i), with differently labeled antihalogenated pyrimidine antibodies which bind specifically to said halogenated monophosphate in each case and so as to provide RNA and DNA with different labels and (iii) separating the differently labeled RNA and DNA.

17. The method of claim 15 wherein said 5-position pyrimidine is 5-bromo uridine and said 5-position halogenated, deoxy pyrimidine is 5-bromo deoxyuridine.

18. The method of claim 15 wherein the label for each of said labeled antibodies is a different member selected from the group consisting of fluorochrome, peroxidase, digoxigen and biotin labels.

19. The method of claim 15 wherein said labeled antibodies are each bound to a solid support.

20. The method of claim 19 wherein said solid support is a member selected from the group consisting of particles, plates, microspheres, heads, magnetized microspheres and fibers.

21. The method of claim 15 which comprises adding a least one enzyme to step (i) which acts to facilitate the incorporation of said 5-position halogenated pyrimidine and said 5-position halogenated deoxy pyrimidine into the RNA and DNA respectively.

16. A method for separating RNA from DNA which comprises the steps of separately incorporating 5-position halogenated uridine triphosphate and 5-position halogenated deoxyuridine triphosphate into the RNA and DNA respectively, by the step of (i) incubating the RNA and DNA with a 5-position halogenated uridine triphosphate and a 5-position halogenated deoxyuridine triphosphate under conditions whereby the incorporation of the 5-position halogenated uridine triphosphate into the RNA and the incorporation of the 5-position halogenated deoxyuridine triphosphate into the DNA can occur, (ii) incubating the RNA and DNA each having incorporated therein a portion of said 5-position halogenated uridine triphosphate and said 5-position halogenated deoxyuridine triphosphate, respectively, the incorporated portion being a monophosphate as formed in step (i), with differently labeled antihalogenated uridine triphosphate antibodies which bind specifically to said halogenated monophosphate in each case and so as to provide RNA and DNA with different labels and (iii) separating the differently labeled RNA and DNA.

22. A method for isolating RNA synthesized during a predetermined period of time by the steps of (i) initiating the incubation of said RNA with a 5-position halogenated pyrimidine under conditions whereby said 5-position halogenated pyrimidine is incorporated into said RNA (ii) interrupting said incubation after a predetermined period of time has elapsed, (iii) incubating the RNA having incorporated therein a portion of said 5-position halogenated pyrimidine, the incorporated portion being a monophosphate as formed in step (i) with an antihalogenated pyrimidine antibody which binds specifically to said halogenated monophosphate and (iv) separating said RNA from said antibody to isolate the RNA synthesized during said predetermined period of time.

24. The method of claim 22 wherein said 5-position halogenated pyrimidine is 5-position brominated uridine.

25. The method of claim 22 wherein said antihalogenated pyrimidine is a labeled antibody.

26. The method of claim 25 wherein the label in said labeled antibody is selected from the group consisting of a fluorochrome label, a peroxidase label, a digoxigen label and a biotin label.

27. The method of claim 22 wherein said antibody is bound to a solid support.

28. The method of claim 27 wherein said solid support is a member selected from the group consisting of particles, plates, microspheres, beads, magnetized microspheres and fibers.

29. The method of claim 22 which comprises adding at least one enzyme to step (1) which acts to facilitate the incorporation of said 5-position halogenated pyrimidine into the RNA.

23. A method for isolating RNA synthesized during a predetermined period of time by the steps of (i) initiating the incubation of said RNA with a 5-position halogenated uridine triphosphate under conditions whereby said 5-position halogenated uridine triphosphate is incorporated into said RNA (ii) interrupting said incubation after a predetermined period of time has elapsed, (iii) incubating the RNA having incorporated therein a portion of said 5-position halogenated uridine triphosphate, the incorporated portion being a monophosphate as formed in step (i) with an antihalogenated pyrimidine antibody which binds specifically to said halogenated monophosphate and (iv) separating said RNA from said antibody to isolate the RNA synthesized during said predetermined period of time.

30. A method for labeling RNA for the purpose of its isolation which comprise the steps of (i) incubating a member selected from the group consisting of prokaryotic cells, eukaryotic cells and virus, with a 5-position halogenated pyrimidine under conditions whereby the 5-position halogenated pyrimidine is incorporated into the RNA of said group member, (ii) incubating the RNA having incorporated therein a portion of said 5-position halogenated pyrimidine, the incorporated portion being a monophosphate as formed in step (i) with an antihalogenated pyrimidine antibody which binds specifically to said halogenated monophosphate providing the labeled RNA (iii) separating the labeled RNA from other components.

31. A method of isolating RNA which comprises the steps of (i) incubating a member selected from the group consisting of prokaryotic cells, eukaryotic cells and viruses, with a 5-position halogenated pyrimidine under conditions whereby the 5-position halogenated pyrimidine is incorporated into the RNA of said group member, (ii) incubating the RNA having incorporated therein a portion of said 5-position halogenated pyrimidine, the incorporated portion being a monophosphate as formed in step (i) with an antihalogenated pyrimidine antibody which binds specifically to said halogenated monophosphate and which is attached to a solid support, (iii) isolating said solid support and (iv) separating the RNA from said antibody to isolate the RNA.

32. A method for separating RNA from DNA which comprises the steps of (i) incubating a member selected from the group consisting of prokaryotic cells, eukaryotic cells or viruses with a 5-position halogenated pyrimidine and a 5-position halogenated deoxy pyrimidine into the RNA and DNA respectively under conditions whereby the incorporation of the 5-position halogenated pyrimidine into the RNA and the incorporation of the 5-position halogenated deoxy pyrimidine into the DNA of said group member can occur, (ii) incubating the RNA and DNA each having incorporated therein a portion of said 5-position halogenated pyrimidine and said 5-position halogenated deoxy pyrimidine, respectively, the incorporated portion being a monophosphate as formed in step (i), with differently labeled antihalogenated pyrimidine antibodies which bind specifically to said halogenated monophosphate in each case and so as to provide RNA and DNA with different labels and (iii) separating the differently labeled RNA and DNA.

33. A method for isolating RNA synthesized during a predetermined period of time which comprises the steps of (i) initiating the incubation of a member selected from the group consisting of prokaryotic cells, eukaryotic cells and viruses with a 5-position halogenated pyrimidine under conditions whereby said 5-position halogenated pyrimidine is incorporated into said RNA of said group member, (ii) interrupting said incubation after a predetermined period of time has elapsed, (iii) incubating the RNA having incorporated therein a portion of said 5-position halogenated pyrimidine, the incorporated portion being a monophosphate as formed in step (i) with an antihalogenated pyrimidine antibody which binds specifically to said halogenated monophosphate and (iv) separating said RNA from said antibody to isolate the RNA synthesized during said predetermined period of time.