Chemical compositions and uses thereof

Patent metadata

Field	Value
Publication number	US-11377689-B2
Application number	US-202117476707-A
Country	US
Kind code	B2
Filing date	Sep 16, 2021
Priority date	Feb 12, 2018
Publication date	Jul 5, 2022
Grant date	Jul 5, 2022

Abstract

The present invention relates to, among other things, probes, compositions, methods, and kits for simultaneous, multiplexed detection and quantification of protein and/or nucleic acid expression in a user-defined region of a tissue, user-defined cell, and/or user-defined subcellular structure within a cell that are adaptable for use with existing sequencing technologies.

First claim

What is claimed is:

1. A method for spatially detecting at least one target analyte in a first location and a second location of a tissue sample comprising:
a) contacting the tissue sample with a plurality of nucleic acid probes, wherein each of the nucleic acid probes comprise a target binding domain that binds to the at least one target analyte, wherein the tissue sample [has been] is treated to facilitate binding of the nucleic acid probes to the target analyte;
b) collecting the nucleic acid probes, or portions thereof, bound to the at least one target analyte in a first location of the tissue sample under conditions that release the nucleic acid probes, or portions thereof, from the first location of the tissue sample;
c) collecting the nucleic acid probes, or portions thereof, bound to the at least one target analyte in a second location of the tissue sample under conditions that release the nucleic acid probes, or portions thereof, from the second location of the tissue sample;
d) performing an extension reaction that incorporates at least one nucleic acid sequence that identifies the first location of the tissue sample into each of the nucleic acid probes, or portions thereof, collected in step (b), thereby forming a first plurality of extension products that comprise the nucleic acid probes, or portions thereof, collected in step (b) and the at least one nucleic acid sequence that identifies the first location of the tissue sample;
e) performing an extension reaction that incorporates at least one nucleic acid sequence that identifies the second location of the tissue sample into each of the nucleic acid probes, or portions thereof, collected in step (c), thereby forming a second plurality extension products that comprise the nucleic acid probes, or portions thereof, collected in step (c) and the at least one nucleic acid sequence that identifies the second location of the tissue sample; and
f) identifying the first plurality of extension products and the second plurality of extension products by sequencing the first plurality of extension products and the second plurality of extension products, thereby spatially detecting the at least one target analyte in the first location of the tissue sample and the second location of the tissue sample.

2. The method of claim 1 , wherein the tissue sample is a formalin-fixed paraffin-embedded (FFPE) tissue sample.

3. The method of claim 1 , wherein the tissue sample is immobilized onto a microscope slide.

4. The method of claim 3 , wherein the microscope slide comprises a plurality of primers immobilized on the microscope slide.

5. The method of claim 4 , wherein the plurality of primers is immobilized on the microscope slide at their 5′ ends.

6. The method of claim 5 , wherein each of step (d) and step (e) comprise performing a solid-phase amplification reaction, wherein the solid-phase amplification reaction is carried out on the microscope slide using the plurality of primers immobilized on the microscope slide.

7. The method of claim 1 , wherein the sequencing step is performed using a next generation sequencing reaction.

8. The method of claim 1 , further comprising amplifying a library using the first plurality of extension products and the second plurality of extension products as templates.

9. The method of claim 1 , wherein the at least one nucleic acid sequence that identifies the first location of the tissue sample comprises at least one unique molecular identifier.

10. The method of claim 1 , wherein the at least one nucleic acid sequence that identifies the second location of the tissue sample comprises at least one unique molecular identifier.

11. The method of claim 1 , wherein the at least one nucleic acid sequence that identifies the first location of the tissue sample comprises at least one amplification primer binding site.

12. The method of claim 1 , wherein the at least one nucleic acid sequence that identifies the second location of the tissue sample comprises at least one amplification primer binding site.

13. The method of claim 1 , wherein the target binding domains comprise a single-stranded nucleic acid molecule.

14. The method of claim 1 , wherein steps (b) and (c) are performed simultaneously.

15. The method of claim 1 , wherein steps (d) and (e) are performed simultaneously.

16. A method for spatially detecting at least one target analyte in a first location and a second location of a tissue sample comprising:
a) contacting the tissue sample with a first plurality of nucleic acid probes and a second plurality of nucleic acid probes,
wherein each of the nucleic acid probes in the first plurality of nucleic acid probes comprise a target binding domain that binds to the at least one target analyte,
wherein each of the nucleic acid probes in the second plurality of nucleic acid probes comprise a target binding domain that binds to the at least one target analyte,
wherein the tissue sample is treated to facilitate binding of the first plurality of nucleic acid probes and the second plurality of nucleic acid probes to the at least one target analyte;

b) forming ligated probes by ligating each of nucleic acid probes from the first plurality of nucleic acid probes and each of nucleic acid probes from the second plurality of nucleic acid probes that are bound to each of identical molecules from the at least one target analyte;
c) collecting the ligated probes, or portions thereof, bound to each of the identical molecules from the at least one target analyte in a first location of the tissue sample under conditions that release the ligated probes, or portions thereof, from the first location of the tissue sample;
d) collecting the ligated probes, or portions thereof, bound to each of the identical molecules from the at least one target analyte in a second location of the tissue sample under conditions that release the probes, or portions thereof, from the second location of the tissue sample;
e) performing an extension reaction that incorporates at least one nucleic acid sequence that identifies the first location of the tissue sample into each of the ligated probes, or portions thereof, collected in step (c), thereby forming a first plurality of extension products that comprise the ligated probes, or portions thereof, collected in step (c) and the at least one nucleic acid sequence that identifies the first location of the tissue sample;
f) performing an extension reaction that incorporates at least one nucleic acid sequence that identifies the second location of the tissue sample into each of the ligated probes, or portions thereof, collected in step (d), thereby forming a second plurality extension products that comprise the ligated probes, or portions thereof, collected in step (d) and the at least one nucleic acid sequence that identifies the at least second location of the tissue sample; and
g) identifying the first plurality of extension products and the second plurality of extension products by sequencing the first plurality of extension products and the second plurality of extension products, thereby spatially detecting the at least one target analyte in the first location of the tissue sample and the second location of the tissue sample.

17. The method of claim 16 , wherein the tissue sample is a formalin-fixed paraffin-embedded (FFPE) tissue sample.

18. The method of claim 16 , wherein the tissue sample is immobilized onto a microscope slide.

19. The method of claim 18 , wherein the microscope slide comprises a plurality of primers immobilized on the mi

Assignees

• Nanostring Technologies Inc

Inventors

• Beechem Joseph M
• Kim Dae
• Hoang Margaret
• Gregory Mark
• Piazza Erin
• Zhou Denise

Classifications

• C12Q2525/191

  incorporating an adaptor · CPC title

• C12Q1/6841

  In situ hybridisation · CPC title

• C12Q1/6858

  Allele-specific amplification · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Q1/6818

  involving interaction of two or more labels, e.g. resonant energy transfer · CPC title