Signal amplification of fluorescence in situ hybridization

• US 9,890,417 B2
• Filed: 11/03/2014
• Issued: 02/13/2018
• Est. Priority Date: 11/03/2014
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Bernick, Kristin, Ach, Robert, Ghosh, Mistuni, Smart, Brian
• Primary Examiner(s): Martinell, James
• Application Number:
  US14/531,563
• Publication Number:
  US 20160122800A1
• Time in Patent Office: 1,198 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 2563/125 the label being enzymatic, i.e. proteins, and non proteins, such as nucleic acid with enzymatic activity
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin

Abstract

Among other things, this disclosure provides a method of detecting a target nucleic acid. Aspects of the method include: (a) obtaining a labeled nucleic acid probe that is complementary to a target nucleic acid, wherein the probe comprises a capture tag; (b) hybridizing the probe with the target nucleic in a fixed cell, in situ, to produce a duplex; (c) linking the probe in the duplex to a peroxidase conjugate via the capture tag to produce a peroxidase-labeled duplex; and (d) incubating the peroxidase-labeled duplex with a peroxidase substrate, wherein the peroxidase activity of the peroxidase conjugate catalyzes deposition of the substrate in the vicinity of the duplex, thereby producing a detectable signal.

Claims

1. A method of detecting a target nucleic acid, the method comprising:

(a) obtaining a labeled nucleic acid probe for a target nucleic acid, wherein the probe is double-stranded and comprises a capture tag in only one strand of the probe, and wherein the one strand comprising the capture tag is complementary to the target nucleic acid;

(b) contacting both strands of the probe, in denatured form, with the target nucleic acid under conditions suitable for nucleic acid hybridization, to produce a duplex comprising the capture tag and a target nucleic acid;

(c) linking the probe in the duplex to a signal amplification conjugate via the capture tag; and

(d) producing an amplified detectable signal from the signal amplification conjugate in the duplex.

2. The method of claim 1, wherein the capture tag is an affinity tag.

3. The method of claim 2, wherein the affinity tag is selected from a hapten and a biotin moiety.

4. The method of claim 1, wherein the capture tag comprises a chemoselective functional group.

5. The method of claim 4, wherein the chemoselective functional group is selected from the group consisting of an alkyne, a cyclooctyne, an azide, a phosphine, a maleimide, a thiol, an alkoxyamine and an aldehyde.

6. The method of claim 1, wherein the labeled nucleic acid probe comprises a PNA, a RNA or a DNA sequence complementary to the target nucleic acid.

7. The method of claim 1, wherein the labeled nucleic acid probe is at least 20 nucleotides in length.

8. The method of claim 1, wherein the target nucleic acid is in a cell.

9. The method of claim 8, wherein the cell is in a tissue section.

10. The method of claim 8, wherein the cell is in a formalin-fixed, paraffin-embedded (FFPE) tissue section.

11. The method of claim 1, wherein:

linking step (c) comprises linking the probe in the duplex to an enzyme conjugate via the capture tag to produce an enzyme-labeled duplex; and

producing step (d) comprises incubating the enzyme-labeled duplex with an enzyme substrate, wherein the enzymatic activity of the enzyme conjugate produces the amplified detectable signal.

12. The method of claim 11, wherein the enzyme substrate comprises a fluorescent compound described by the formula:

(R¹)_n—

(X)_q—

(R²)_m

wherein each R¹ is independently, a phenol-containing group, a ferulic acid, a cinnamic acid or a cinnapinic acid group;

each R² is a fluorophore;

each X is a linker group or a chemical bond; and

m, n and q are each independently an integer from 1 to 10.

13. The method of claim 12, wherein the fluorescent compound is Fer-4-Flu.

14. The method of claim 11, wherein the enzyme substrate comprises a fluorescent compound and a crosslinker capable of peroxidase-catalyzed radical chain polymerization to produce an insoluble adduct.

15. The method of claim 14, wherein the crosslinker is selected from 3,3′

-diamino benzidin (DAB) and alpha-cyano-4-hydroxy-cinnamic acid (α

-CHC).

16. The method of claim 11, wherein the enzyme conjugate is a peroxidase conjugate and the enzyme substrate is a peroxidase substrate.

17. The method of claim 16, wherein the peroxidase conjugate comprises an HRP polymer.

18. The method of claim 17, wherein the peroxidase conjugate is an HRP-azide conjugate or an HRP-cyclooctyne conjugate.

19. The method of claim 17, wherein the peroxidase enzyme is horse radish peroxidase (HRP) or soybean peroxidase (SP).

20. The method of claim 1, wherein the target nucleic acid is an RNA transcript.