In-situ hybridization method using RecA protein and RecA protein having marker or ligand for use in said method

• US 5,965,361 A
• Filed: 10/30/1995
• Issued: 10/12/1999
• Est. Priority Date: 12/28/1993
• Status: Expired due to Fees

• Current Assignee: TAPESTRY PHARACEUTICALS, INC.
• Original Assignee: Daikin Industries Ltd
• Inventors: Mukai, Eli, Kihara, Kayo, Kigawa, Koji, Yamanaka, Mikayo, Obata, Kazuaki
• Primary Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US08/507,423
• Time in Patent Office: 1,443 Days
• Field of Search: 530/350, 530/387.1, 935/77, 935/78, 935/76, 435/4, 435/6, 435/7.1, 435/40.5, 435/172.3, 435/810, 536/24.3, 536/24.31, 536/24.32
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

A method for detecting the presence of a double-stranded target nucleic acid sequence contained in fixed cells or cell structures is provided. The method includes the steps of forming the fixed cells or cell structures by fixing cells or cell structures so as to allow a nucleic acid probe to enter, and forming a probe/RecA complex in which a single-stranded probe and RecA protein are stably bound to each other. The probe/RecA complex is allowed to react with the double-stranded target nucleic acid sequence to bind thereto under conditions in which the double-stranded target nucleic acid sequence is not denatured, and by detecting the RecA protein included in the probe/RecA complex, the presence of the double-stranded target nucleic acid sequence is detected.

The invention provides a diagnostic method by which the position of a specific gene or its regulatory region in a chromosome and the presence of a nucleic acid sequence derived from virus can be measured or visualized with high sensitivity and with ease.

Claims

1. A method for detecting the presence of a double-stranded target nucleic acid sequence contained in fixed cells or cell structures, the method comprising the steps of:

obtaining the fixed cells or cell structures by fixing cells or cell structures so as to allow a nucleic acid probe to enter;

forming a probe/labeled RecA complex in which a single-stranded probe and RecA protein having a detectable label or ligand are stably bound to each other, the single-stranded probe having a sequence complementary to the double-stranded target nucleic acid sequence;

adding the probe/labeled RecA complex to the fixed cells or cell structures under conditions in which the probe/labeled RecA complex can come in contact with the double-stranded target nucleic acid sequence;

allowing the probe/labeled RecA complex to react with the double-stranded target nucleic acid sequence so as to bind thereto under conditions where the double-stranded target nucleic acid sequence is not denatured;

removing the unreacted part of the probe/labeled RecA complex that has not been bound to the double-stranded target nucleic acid sequence; and

detecting the presence of the double-stranded target nucleic acid sequence by detecting the RecA protein having a detectable label or ligand included in the probe/labeled RecA complex bound to the double-stranded target nucleic acid.

2. A method according to claim 1,

wherein the label or ligand is selected from the group consisting of a fluorescent indicator, a chemiluminescent agent, an enzymatic label, a radioactive label, biotin and digoxigenin.

3. A method according to claim 1,

wherein the step of forming the probe/RecA complex is performed in the presence of a cofactor selected from the group consisting of ATPγ

S, GTPγ

S, rATP, DATP, a mixture of ATPγ

S and rATP, and a mixture of ATPγ

S and ADP.

4. A method according to claim 2,

wherein the fluorescent indicator is selected from the group consisting of fluoroscein isothiocyanate, carboxymethylindocyanine succinimidyl ester, rhodamine, Texas red (sulforhodamine), tetramethylrhodamine isothiocyanate, and 7-amino-4-methylcoumarin-3-acetic acid.

5. A method according to claim 1,

wherein the label is a fluorescent indicator, and the step of detecting the presence of the double-stranded target nucleic acid sequence is performed with a fluorescence microscope or a flow cytometer.

6. A method according to claim 2,

wherein the enzymatic label is selected from the group consisting of alkaline phosphatase, peroxidase and β

-galactosidase.

7. A method according to claim 1,

wherein the step of detecting the presence of the double-stranded target nucleic acid sequence is performed by further using an anti-RecA antibody.

8. A method according to claim 7,

wherein the anti-RecA antibody has a detectable label or ligand.

9. A method according to claim 8,

wherein the label or ligand is selected from the group consisting of a fluorescent indicator, a chemiluminescent agent, an enzymatic label, a radioactive label, biotin and digoxigenin.

10. A method according to claim 9,

wherein the fluorescent indicator is selected from the group consisting of fluoroscein isothiocyanate, carboxymethylindocyanine succinimidyl ester, rhodamine, Texas red (sulforhodamine), tetramethylrhodamine isothiocyanate, and 7-amino-4-methylcoumarin-3-acetic acid.

11. A method according to claim 9,

wherein the label is a fluorescent indicator, and the step of detecting the presence of the double-stranded target nucleic acid sequence is performed with a fluorescence microscope or a flow cytometer.

12. A method according to claim 9,

wherein the enzymatic label is selected from the group consisting of alkaline phosphatase, peroxidase and β

-galactosidase.

13. A method according to claim 1,

wherein the step of detecting the presence of the double-stranded target nucleic acid sequence is performed by further using an anti-RecA antibody and a secondary antibody having a detectable label or ligand that can bind to an anti-RecA antibody.

33. A method according to claim 1, further comprising a step of treating the fixed cells or cell structures with a pre-blocking solution in order to prevent the non-specific binding of the RecA protein having a detectable label or ligand to a component in the fixed cells or cell structures.

34. A method according to claim 33,

wherein the pre-blocking solution includes at least one selected from the group consisting of casein, skim milk, bovine serum albumin, carrier DNA and carrier RNA that is hybridized with neither nucleic acid contained in the cells or cell structures nor the probe.

14. A kit including RecA protein having a detectable label or ligand, for use in practicing a method for detecting the presence of a double-stranded target nucleic acid sequence contained in fixed cells or cell structures, the method comprising the steps of:

obtaining the fixed cells or cell structures by fixing cells or cell structures so as to allow a nucleic acid probe to enter;

forming a probe/labeled RecA complex in which a single stranded probe and RecA protein having a detectable label or ligand are stably bound to each other, the single-stranded probe having a sequence complementary to the double-stranded target nucleic acid sequence;

adding the probe/labeled RecA complex to the fixed cells or cell structures under conditions in which the probe/labeled RecA complex can come in contact with the double-stranded target nucleic acid sequence;

allowing the probe/labeled RecA complex to react with the double-stranded target nucleic acid sequence so as to bind thereto under conditions where the double-stranded target nucleic acid sequence is not denatured;

removing the unreacted part of the probe/labeled RecA complex that has not been bound to the double-stranded target nucleic acid sequence; and

detecting the presence of the double-stranded target nucleic acid sequence by detecting the RecA protein having a detectable label or ligand included in the probe/labeled RecA complex bound to the double-stranded target nucleic acid.

15. A kit according to claim 14,

wherein the label or ligand is selected from the group consisting of a fluorescent indicator, a chemiluminescent agent, an enzymatic label, a radioactive label, biotin and digoxigenin.

16. A kit according to claim 15,

wherein the fluorescent indicator is selected from the group consisting of fluoroscein isothiocyanate, carboxymethylindocyanine succinimidyl ester, rhodamine, Texas red (sulforhodamine), tetramethylrhodamine isothiocyanate, and 7-amino-4-methylcoumarin-3-acetic acid.

17. A kit according to claim 15,

wherein the enzymatic label is selected from the group consisting of alkaline phosphatase, peroxidase and β

-galactosidase.

18. A kit including probe/labeled RecA complex in which a probe and RecA protein having a detectable label or ligand are stably bound to each other, for use in practicing a method for detecting the presence of a double-stranded target nucleic acid sequence contained in fixed cells or cell structures, the method comprising the steps of:

obtaining the fixed cells or cell structures by fixing cells or cell structures so as to allow a nucleic acid probe to enter;

forming a probe/labeled RecA complex in which a single-stranded probe and RecA protein having a detectable label or ligand are stably bound to each other, the single-stranded probe having a sequence complementary to the double-stranded target nucleic acid sequence,

adding the probe/labeled RecA complex to the fixed cells or cell structures under conditions in which the probe/labeled RecA complex can come in contact with the double stranded target nucleic acid sequence;

allowing the probe/labeled RecA complex to react with the double-stranded target nucleic acid sequence so as to bind thereto under conditions where the double-stranded target nucleic acid sequence is not denatured;

removing the unreacted part of the probe/labeled RecA complex that has not been bound to the double-stranded target nucleic acid sequence; and

detecting the presence of the double-stranded target nucleic acid sequence by detecting the RecA protein having a detectable label or ligand included in the probe/labeled RecA complex bound to the double-stranded target nucleic acid.

19. A kit according to claim 18,

wherein the label or ligand is selected from the group consisting of a fluorescent indicator, a chemiluminescent agent, an enzymatic label, a radioactive label, biotin and digoxigenin.

20. A kit according to claim 19,

wherein the fluorescent indicator is selected from the group consisting of fluoroscein isothiocyanate, carboxymethylindocyanine succinimidyl ester, rhodamine, Texas red (sulforhodamine), tetramethylrhodamine isothiocyanate, and 7-amino-4-methylcoumarin-3-acetic acid.

21. A kit according to claim 19,

wherein the enzymatic label is selected from the group consisting of alkaline phosphatase, peroxidase and β

-galactosidase.

22. A kit including RecA protein and an anti-RecA antibody comprising:

RecA protein and an anti-RecA antibody both having a detectable label or ligand,

wherein the kit is used for practicing a method for detecting the presence of a double-stranded target nucleic acid sequence contained in fixed cells or cell structures, the method comprising the steps of;

obtaining the fixed cells or cell structures by fixing cells or cell structures so as to allow a nucleic acid probe to enter;

forming a probe/labeled RecA complex in which a single-stranded probe and RecA protein having a detectable label or ligand are stably bound to each other, the single-stranded probe having a sequence complementary to the double-stranded target nucleic acid sequence;

adding the probe/labeled RecA complex to the fixed cells or cell structures under conditions in which the probe/labeled RecA complex can come in contact with the double-stranded target nucleic acid sequence;

allowing the probe/labeled RecA complex to react with the double-stranded target nucleic acid sequence so as to bind thereto under conditions where the double-stranded target nucleic acid sequence is not denatured;

removing the unreacted part of the probe/labeled RecA complex that has not been bound to the double-stranded target nucleic acid sequence; and

detecting the presence of the double-stranded target nucleic acid sequence by detecting the RecA protein having a detectable label or ligand included in the probe/labeled RecA complex bound to the double-stranded target nucleic acid,

wherein the step of detecting the presence of the double-stranded target nucleic acid sequence is performed by further using an anti-RecA antibody having a detectable label or ligand.

23. A kit according to claim 22,

wherein the label or ligand is selected from the group consisting of a fluorescent indicator, a chemiluminescent agent, an enzymatic label, a radioactive label , biotin and digoxigenin.

24. A kit according to claim 23,

wherein the fluorescent indicator is selected from the group consisting of fluoroscein isothiocyanate, carboxymethylindocyanine succinimidyl ester, rhodamine, Texas red (sulforhodamine), tetramethylrhodamine isothiocyanate, and 7-amino-4-methylcoumarin-3-acetic acid.

25. A kit according to claim 23,

wherein the enzymatic label is selected from the group consisting of alkaline phosphatase, peroxidase and β

-galactosidase.

26. A kit including a probe/labeled RecA complex comprising:

RecA protein and an anti-RecA antibody both having a detectable label or ligand,

wherein the RecA protein, having the detectable label or ligand, and a probe are stably bound to each other, for use in practicing a method for detecting the presence of a double-stranded target nucleic acid sequence contained in fixed cells or cell structures, the method comprising the steps of;

obtaining the fixed cells or cell structures by fixing cells or cell structures so as to allow a nucleic acid probe to enter;

forming a probe/labeled RecA complex in which a single-stranded probe and RecA protein having a detectable label or ligand are stably bound to each other, the single-stranded probe having a sequence complementary to the double-stranded target nucleic acid sequence;

adding the probe/labeled RecA complex to the fixed cells or cell structures under conditions in which the probe/labeled RecA complex can come in contact with the double-stranded target nucleic acid sequence;

allowing the probe/labeled RecA complex to react with the double-stranded target nucleic acid sequence so as to bind thereto under conditions where the double-stranded target nucleic acid sequence is not denatured;

removing the unreacted part of the probe/labeled RecA complex that has not been bound to the double-stranded target nucleic acid sequence; and

detecting the presence of the double-stranded target nucleic acid sequence by detecting the RecA protein having a detectable label or ligand included in the probe/labeled RecA complex bound to the double-stranded target nucleic acid,

wherein the step of detecting the presence of the double-stranded target nucleic acid sequence is performed by further using anti-RecA antibody having a detectable label or ligand.

27. A kit according to claim 26,

wherein the label or ligand is selected from the group consisting of a fluorescent indicator, a chemiluminescent agent, an enzymatic label, a radioactive label, biotin and digoxigenin.

28. A kit according to claim 27,

wherein the fluorescent indicator is selected from the group consisting of fluoroscein isothiocyanate, carboxymethylindocyanine succinimidyl ester, rhodamine, Texas red (sulforhodamine), tetramethylrhodamine isothiocyanate, and 7-amino-4-methylcoumarin-3-acetic acid.

29. A kit according to claim 27,

wherein the enzymatic label is selected from the group consisting of alkaline phosphatase, peroxidase and β

-galactosidase.

30. RecA protein

having a label or ligand selected from the group consisting of an enzymatic label, biotin and digoxigenin.

31. RecA protein according to claim 30 comprising, per RecA protein monomer, 1 to 3 molecules of an enzymatic label selected from the group consisting of alkaline phosphatase, peroxidase and β

-galactosidase.

32. RecA protein according to claim 30 comprising, per RecA protein monomer, 1 to 6 molecules of a ligand selected from the group consisting of biotin and digoxigenin.