IN SITU HYBRIDIZATION DETECTION METHOD

• US 20160273035A1
• Filed: 03/17/2015
• Published: 09/22/2016
• Est. Priority Date: 03/17/2015
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Dako Denmark A/S (Agilent Technologies, Inc.)
• Inventors: Winther, Lars, Lohse, Jesper, Gabs, Susanne, Petersen, Kenneth Heesche
• Granted Patent: US 10,036,066 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2565/40 Detection characterised by signal amplification of label

Abstract

The invention provides compositions and methods for the detection of targets in a sample; in particular, an in situ hybridization (ISH) sample. Probes and detectable labels may be provided in multiple layers in order to increase the flexibility of a detection system, and to allow for amplification to enhance the signal from a target. The layers may be created by incorporating probes and detectable labels into larger molecular units that interact through nucleic acids base-pairing, including peptide-nucleic acid (PNA) base-pairing. Optional non-natural bases allow for degenerate base pairing schemes. The compositions and methods are also compatible with immunohistochemistry (IHC), immunocytochemistry (ICC), flow cytometry, enzyme immuno-assays (EIA), enzyme linked immuno-assays (ELISA), blotting methods (e.g. Western, Southern, and Northern), labeling inside electrophoresis systems or on surfaces or arrays, and precipitation, among other general detection assay formats. The invention is also compatible with many different types of targets, probes, and detectable labels.

Claims

1. A composition comprising at least one recognition unit and at least one detection unit wherein:

a) each unit independently comprises at least one nucleic acid analog segment;

b) at least one nucleic acid analog segment of the recognition unit specifically hybridizes to at least one nucleic acid analog segment of the detection unit;

c) the recognition unit further comprises at least one probe which recognizes at least one target in a sample;

d) the detection unit further comprises at least one detectable label; and

e) the at least one nucleic acid analog segments on the recognition unit and detection unit that specifically hybridize to other nucleic acid analog segments on the recognition unit and detection unit do not specifically hybridize to the probe, detectable label, or target; and

wherein the sample is an in situ hybridization (ISH) sample.

2. The composition according to claim 1, wherein at least one nucleic acid analog segment comprises at least one non-natural base.

3. The composition according to claim 2, wherein the non-natural base is chosen from xanthine, a thio-xanthine, inosine, diaminopurine, pyrimidinone, a thiouracil, 2-thio-guanine, and 2-amino-6-thioxopurine.

4. The composition according to claim 2, wherein at least one nucleic acid analog segment comprises a peptide-nucleic acid (PNA) backbone.

5. The composition of claim 1, wherein the probe is a nucleic acid or nucleic acid analog segment.

6. The composition according to claim 5, wherein the probe is a nucleic acid analog segment that comprises at least one non-natural base.

7. The composition according to claim 6, wherein the at least one non-natural base is chosen from xanthine, a thio-xanthine, inosine, diaminopurine, pyrimidinone, a thio-uracil, 2-thio-guanine, and 2-amino-6-thioxopurine.

8. The composition according to claim 5, wherein the probe is a nucleic acid analog segment that comprises a peptide-nucleic acid (PNA) backbone.

9. The composition according to claim 1, wherein each unit comprises at least two nucleic acid analog segments that specifically hybridize to at least one other unit but do not specifically hybridize to the probe, detectable label, or target;

and wherein said at least two nucleic acid analog segments optionally comprise different nucleobase sequences.

10. The composition according to claim 1, wherein at least one nucleic acid analog segment on one of the units is capable of specifically hybridizing to at least two nucleic acid analog segments on another unit, each comprising a different nucleobase sequence.

11. The composition according to claim 1, wherein the probe recognizes the target indirectly by binding to a primary, secondary, or tertiary binding agent.

12. The composition according to claim 1, wherein at least one unit further comprises at least one polymer.

13. The composition according to claim 12, wherein the polymer is dextran.

14. The composition according to claim 1, wherein at least one unit further comprises at least one linker.

15. The composition according to claim 14, wherein the linker is chosen from a molecule comprising polyethylene glycol and a molecule comprising at least two units according to the Formula I:

69. A method of detecting a target in a sample comprising:

contacting the composition of claim 1 with a sample comprising the target such that the probe of the composition of claim 1 hybridizes to the target;

detecting a signal for the detectable label of the composition of claim 1, thereby detecting the target in the sample.

70. The method of claim 69, further comprising comparing the signal from the target in the sample with the signal from a reference target or reference sample.

71. The method of claim 69, wherein more than one detection unit is associated with each recognition unit of the composition of claim 1.

72. The method of claim 69, wherein at least two targets are detected in the sample.

73. The method of claim 69, wherein each unit of the composition of claim 1 comprises at least two nucleic acid analog segments.

16-68. -68. (canceled)