Multiplexed in situ molecular analyses and programmable molecular probes for regulated single amplification

Patent metadata

Field	Value
Publication number	US-10150988-B2
Application number	US-201314421504-A
Country	US
Kind code	B2
Filing date	Aug 13, 2013
Priority date	Aug 13, 2012
Publication date	Dec 11, 2018
Grant date	Dec 11, 2018

Abstract

The present invention generally relates to methods for detecting a target in a sample; methods for modulating the reporting intensity of a labeled target in a sample of fixed cells or tissues; methods for detecting the location of at least two targets in a sample; and related compositions.

First claim

What is claimed:

1. A method for modulating the reporting intensity of a labeled target in a sample of fixed cells or tissues, the method comprising:
complexing a target-recognition agent to the target, wherein the target-recognition agent comprises a first polynucleotide, to form a first polynucleotide-target complex;
reacting the first polynucleotide-target complex via strand displacement with a branched or dendritic dynamic DNA complex to create a dynamic DNA-target complex;
reacting the dynamic DNA-target complex via strand displacement with a first probe comprising at least one dye and a second polynucleotide to form a first labeling complex;
measuring the reporting intensity of the first labeling complex; and
contacting the sample with a second probe comprising at least one dye and a third polynucleotide, wherein the second probe displaces the first probe via strand displacement to form a second labeling complex;
wherein the second probe carries a different number and/or type of dye molecule than the first probe.

2. The method of claim 1 , wherein at least one dye is a fluorescent dye.

3. The method of claim 1 , where at least one probe comprises at least two dye molecules.

4. The method of claim 3 , wherein the at least two dye molecules are the same dye.

5. The method of claim 3 , wherein the at least two dye molecules are different dyes.

6. The method of claim 1 , wherein the first probe and the second probe comprise different dyes.

7. The method of claim 1 , wherein the first labeling complex and the second labeling complex have a 36:1 to 1:36 ratio of dye molecules.

8. The method of claim 1 , wherein the first labeling complex and the second labeling complex have a 9:1 to 1:9 ratio of dye molecules.

9. The method of claim 1 , wherein at least one polynucleotide is at least one selected from a group consisting of DNA and RNA.

10. The method of claim 1 , wherein the target-recognition agent comprises at least one antibody, peptide, aptamer, enzyme, or self-assembling protein.

11. The method of claim 1 , wherein the first polynucleotide comprises a region that binds to the target.

12. The method of claim 1 , further comprising measuring the reporting intensity of the second labeling complex.

Assignees

• Univ Rice William M

Inventors

• Diehl Michael
• Zimak Jan
• Schweller Ryan
• Samson Edward B
• Duose Dzifa Y

Classifications

• C12Q1/6816

  characterised by the detection means (C12Q1/6804 takes precedence) · CPC title

• C12Q1/6841

  In situ hybridisation · CPC title

• C12Q1/682 Primary

  Signal amplification · CPC title

• C12Q2531/119

  Strand displacement amplification [SDA] · CPC title

• C12Q2525/313

  Branched oligonucleotides · CPC title