Compositions and methods for analyte detection

Patent metadata

Field	Value
Publication number	US-12559790-B2
Application number	US-202217875211-A
Country	US
Kind code	B2
Filing date	Jul 27, 2022
Priority date	Jan 29, 2020
Publication date	Feb 24, 2026
Grant date	Feb 24, 2026

Abstract

The present disclosure provides methods, systems and compositions for detecting nucleic acid sequences in a biological sample having a three-dimensional matrix. The present disclosure also provides methods, systems and compositions for processing a biological sample for use in nucleic acid sequence detection.

First claim

What is claimed is:

1 . A method for processing a biological sample, comprising:
(a) providing said biological sample comprising: (i) a polypeptide analyte and (ii) a nucleic acid analyte; (b) contacting said biological sample with a tethering molecule, thereby binding said tethering molecule to said polypeptide analyte, wherein said tethering molecule comprises; (i) an antibody or fragment thereof that binds to said polypeptide analyte; (ii) a nucleic acid barcode conjugated to said antibody or fragment thereof, wherein said nucleic acid barcode identifies said antibody or fragment thereof; and (iii) a tethering moiety; and (c) contacting said nucleic acid analyte with a primer sequence, wherein said primer sequence hybridizes to a sequence of said nucleic acid analyte; (d) extending said primer sequence hybridized to said sequence of said nucleic acid analyte to generate an extension product; (e) contacting said tethering molecule bound to said polypeptide analyte with a matrix-forming material comprising an attachment moiety, and coupling said tethering moiety of said tethering molecule to said attachment moiety of said matrix-forming material, thereby generating a synthetic 3D matrix that is attached to said polypeptide analyte; (f) hybridizing a probe to said extension product; and (g) subjecting said probe or derivative thereof to an amplification reaction using a modified base to generate an amplification product comprising said modified base.

2 . The method of claim 1 , wherein said synthetic 3D matrix comprises an additional attachment moiety.

3 . The method of claim 2 , wherein said attachment moiety and said additional attachment moiety are different.

4 . The method of claim 2 , wherein said primer sequence (i) flows through said synthetic 3D matrix and comes in contact with said nucleic acid analyte, and (ii) attaches to said nucleic acid analyte and said additional attachment moiety of said synthetic 3D matrix, thereby attaching said nucleic acid analyte to said synthetic 3D matrix.

5 . The method of claim 1 , further comprising contacting said biological sample with said tethering molecule and said matrix-forming material concurrently.

6 . The method of claim 1 , wherein said nucleic acid analyte is an RNA analyte, and wherein said extension product comprises complementary deoxyribonucleic acid (cDNA).

7 . The method of claim 6 , further comprising releasing said cDNA from said RNA analyte.

8 . The method of claim 1 , wherein said extension product is attached to said synthetic 3D matrix.

9 . The method of claim 1 , wherein said probe is a padlock probe or a molecular inversion probe.

10 . The method of claim 1 , further comprising circularizing said probe.

11 . The method of claim 10 , wherein said circularizing comprises ligating a 3′ end and a 5′ end of said probe.

12 . The method of claim 1 , wherein said amplification reaction is rolling circle amplification.

13 . The method of claim 1 , wherein said modified base is 5-azidomethyl-dUTP.

14 . The method of claim 1 , wherein said tethering moiety comprises an acryloyl group.

15 . The method of claim 1 , further comprising contacting said biological sample with said tethering molecule and said matrix-forming material sequentially.

16 . The method of claim 1 , further comprising subjecting said biological sample to a clearing process.

17 . The method of claim 1 , further comprising attaching said amplification product to said synthetic 3D matrix.

18 . The method of claim 17 , further comprising attaching said amplification product to said synthetic 3D matrix via a click chemistry reaction.

19 . The method of claim 1 , further comprising detecting said nucleic acid barcode to identify said antibody or fragment thereof, thereby identifying said polypeptide analyte in said biological sample.

20 . The method of claim 1 , further comprising detecting said amplification product to identify said nucleic acid analyte in said biological sample.

Assignees

• 10X Genomics Inc

Inventors

• Daugharthy Evan
• Terry Richard
• Camplisson Conor
• Barclay David
• Miselis Tyler
• Chen Hong
• Droz Shoshoni

Classifications

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Q1/6804

  Nucleic acid analysis using immunogens (immunoassay G01N33/53) · CPC title

• C12Q1/6841 Primary

  In situ hybridisation · CPC title