In situ hybridization method

• US 5,719,023 A
• Filed: 06/03/1994
• Issued: 02/17/1998
• Est. Priority Date: 09/04/1991
• Status: Expired due to Fees

• Current Assignee: TAPESTRY PHARACEUTICALS, INC.
• Original Assignee: Daikin Industries Ltd
• Inventors: Sena, Elissa P., Calhoun, Cornelia J., Zarling, David A.
• Primary Examiner(s): Campbell, Eggerton A.
• Application Number:
  US08/199,326
• Time in Patent Office: 1,355 Days
• Field of Search: 435/5, 435/6, 935/77, 935/78
• US Class Current: 435/6.16
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• 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: C12Q 1/706 for hepatitis
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

A method of identifying the presence of a known target sequence in nucleic acid contained in a fixed cellular or subcellular biological structure. By adding a stable, reporter-labeled RecA/single-stranded probe complex to the cellular or subcellular structure, the target sequence can be effectively labeled by in situ hybridization, allowing the target sequence to be visualized histologically and microscopically or detected by in situ cytometry or cell sorting flow techniques.

Claims

1. A method of identifying the presence of a known target sequence in a double-stranded nucleic acid contained in a fixed cellular or subcellular biological structure, in a defined morphological relationship with the structure, comprising

adding to the structure, a probe complex composed of RecA protein stably bound to a single-stranded, reporter-labeled probe which is complementary to a duplex target sequence, under conditions in which the complex can contact the duplex nucleic acid target,

allowing the complex to bind to the target sequence under non-denaturing conditions,

removing unbound complex from said structure, and

examining the structure for the presence of the reporter-labeled probe bound to the nucleic acid.

2. The method of claim 1, wherein the complex is stabilized by the presence of a cofactor selected from the group consisting of ATPγ

S, GTPγ

S, ATP, dATP and a combination of ATPγ

S and ADP.

3. The method of claim 1, wherein said probe is labeled with a ligand reporter, and said examining includes adding to the structure, specific ligand molecule, including antibodies, effective to stably bind to said ligand, and having a detectable reporter group.

4. The method of claim 1, for detecting the presence in a host cell, of a pathogenic (foreign) target duplex nucleic acid sequence, wherein said complex is added to the cells under conditions of host cell fixation, and said examining includes detecting the presence of a probe-bound reporter in said fixed cells.

5. The method of claim 1, wherein said examining includes detecting a fluorescent reporter bound to the reporter-labeled probe bound to the nucleic acid using either microscopy or a fluorescence activated cell sorter.

6. The method of claim 1, for localizing a selected target duplex nucleic acid sequence integrated into a host-cell genome, wherein said complex is added to the chromosomes of the cell, and said examining includes examining the chromosomes microscopically to determine the relative position of reporter-labeled probe in relation to chromosome ultrastructure.

7. The method of claim 6, wherein said chromosomes are labeled with one fluorescence reporter, said probe is labeled with a second fluorescence reporter, and said examining includes viewing the cells by fluorescence microscopy separately at wavelengths effective to excite fluorescence in each of the two reporters.

8. The method of claim 6, for localizing the target sequence in a selected chromosome, which further includes adding to the structure a second probe complex composed of RecA protein stably bound to a single-stranded, reporter-labeled nucleic acid probe which is complementary to a duplex strand in a known region of the selected chromosome, and said examining includes determining the relative positions of reporters associated with each of the two complexes.

9. The method of claim 8, wherein the first-mentioned complex and the second complex are labeled with different fluorescence reporters, and said examining includes viewing the cells by fluorescence microscopy separately at wavelengths effective to excite fluorescence in each of the two reporters.

10. The method of claim 1, which further includes amplifying the target duplex nucleic acid in the structure prior to said adding.

11. The method of claim 1, which further includes amplifying the probe bound to the target by addition of polymerase, and all four deoxytrinucleotides, where one of the deoxytrinucleotides includes a reporter label.

12. The method of claim 1, where said fixed structures are in solution or on a slide.

13. The method of claim 1, where said fixed structures are incubated in 10 mM Tris-acetate buffer, pH 7.5, at 55°

-60°

C. before the addition of said RecA probe complex.

14. The method of claim 1, where said allowing the complex to bind to the target sequence under non-denaturing conditions is carried out for less than 2 hours.

15. The method of claim 1, where said adding includes the addition of accessory proteins selected from the group consisting of toposiomerase I and topoisomerase II.

16. A method of identifying the presence of a known double-stranded viral nucleic acid target sequence contained in a fixed cellular or subcellular biological structure, comprising

adding to the structure, a prone complex composed of RecA protein stably bound to a single-stranded, reporter-labeled probe which is complementary to the double-stranded viral nucleic acid target sequence, under conditions in which the complex can contact the double-stranded nucleic acid target,

allowing the complex to bind the target sequence under non-denaturing conditions,

removing unbound complex from said structure, and

examining the structure for the presence of the reporter-labeled probe bound to the nucleic acid.

17. The method of claim 16, where the known viral target is a sequence derived from hepatitis B virus.

18. The method of claim 16, where the fixed structures are incubated in 10 mM Tris-acetate buffer, pH 7.5, at 55°

-60°

C. before the addition of the RecA probe complex.

19. The method of claim 16, wherein the complex is stabilized by the presence of a cofactor selected from the group consisting of ATP S, GTP S, ATP, dATP and a combination of ATP S and ADP.

20. The method of claim 16, wherein said probe is labeled with a ligand reporter, and said examining includes adding to the structure, specific ligand molecule, effective to stably bind to said ligand, and having a detectable reporter group.

21. The method of claim 20, wherein said ligand reporter is digoxigenin or biotin and said ligand molecule is selected from the group consisting of an antibody, avidin and streptavidin.

22. The method of claim 16, wherein said examining includes detecting a fluorescent reporter bound to the reporter-labeled probe bound to the nucleic acid using either microscopy or a fluorescence activated cell sorter.

23. The method of claim 16, for localizing a selected target duplex nucleic acid sequence integrated into a host-cell genome, wherein said complex is added to the chromosomes of the cell, and said examining includes examining the chromosomes microscopically to determine the relative position of reporter-labeled probe in relation to chromosome ultrastructure.

24. A kit for the practice of the method of claim 16, comprising a RecA-protein coated single-stranded and reporter labelled DNA probe derived from the viral nucleic acid sequences, means of removing unbound complex from said structure, and means of examining the structure for the presence of the reporter labelled probe bound to the nucleic acid.

25. The kit of claim 24, where the probe is derived from hepatitis B virus sequences.

26. The kit of claim 24, where the reporter is biotin or digoxigenin.

27. The kit of claim 24, where the kit further includes means of detecting the binding of the probe to the known double-stranded viral nucleic acid sequences in a sample and said means of detection includes detecting a fluorescent reporter bound to the reporter-labeled probe bound to the nucleic acid using either microscopy or a fluorescence activated cell sorter.

28. The method of claim 16, where said fixed structures are in solution or on a slide.

29. The method of claim 16, where said allowing the complex to bind to the target sequence under non-denaturing conditions is carried out for less than 2 hours.

30. The method of claim 16, where said adding includes the addition of accessory proteins selected from the group consisting of toposiomerase I and topoisomerase II.

31. A method of detecting a single copy nucleic acid target sequence contained in a cellular or subcellular biological structure, comprising

fixing the cellular or subcellular biological structure,

adding to the structure, a probe complex composed of RecA protein stably bound to a single-stranded, reporter-labeled probe which is complementary to the single-copy nucleic acid target sequence, under conditions in which the complex can contact the nucleic acid target,

allowing the complex to bind to the target sequence under non-denaturing conditions,

removing unbound complex from said structure, and

examining the structure for the presence of the reporter-labeled probe bound to the nucleic acid.

32. The method of claim 31, where said fixing is in solution or on a slide.

33. The method of claim 31, where said fixing includes incubation of the fixed structures in 10 mM Tris-acetate buffer, pH7.5, at 55°

-60°

C.

34. The method of claim 31, where said allowing the complex to bind to the target sequence under non-denaturing conditions is carried out for less than 2 hours.

35. The method of claim 31, where said adding includes the addition of accessory proteins selected from the group consisting of toposiomerase I and topoisomerase II.

36. The method of claim 31, wherein the complex is stabilized by the presence of a cofactor selected from the group consisting of ATPγ

S, GTPγ

S, ATP, dATP, and a combination of ATPγ

S and ADP.

37. The method of claim 31, wherein said probe is labeled with a ligand reporter, and said examining includes adding to the structure, specific ligand molecule, effective to stably bind to said ligand, and having a detectable reporter group.

38. The method of claim 37, wherein said ligand reporter is digoxigenin or biotin and said ligand molecule is selected from the group consisting of an antibody, avidin and streptavidin.

39. The method of claim 31, wherein said examining includes detecting a fluorescent reporter bound to the reporter-labeled probe bound to the nucleic acid using either microscopy or a fluorescence activated cell sorter.

40. The method of claim 31, for localizing a selected target duplex nucleic acid sequence integrated into a host-cell genome, wherein said complex is added to the chromosomes of the cell, and said examining includes examining the chromosomes microscopically to determine the relative position of reporter-labeled probe in relation to chromosome ultrastructure.

41. A kit for the practice of the method of claim 34, comprising a RecA-protein coated single-stranded and reporter labelled DNA probe derived from the single copy nucleic acid sequences, means of removing unbound complex from said structure, and means of examining the structure for the presence of the reporter labelled probe bound to the nucleic acid.

42. The kit of claim 41, where the probe is derived from p53 tumor suppressor gene sequences.

43. The kit of claim 41, where the reporter is biotin or digoxigenin.

44. The kit of claim 41, where the kit further includes means of detecting the binding of the probe to the single copy nucleic acid sequences in a sample and said means of detection includes detecting a fluorescent reporter bound to the reporter-labeled probe bound to the nucleic acid using either microscopy or a fluorescence activated cell sorter.