IP Library › Granted Patent US 12,319,955
Granted Patent B2
US 12,319,955 · App. 18/343,858 · Granted Jun 3, 2025

Materials and methods for localized detection of nucleic acids in a tissue sample

Inventor: Jun Hee Lee (Ann Arbor, MI)
Assignee: The Regents of the University of Michigan
C12Q1/6806C12N15/1065C12Q1/6841C12Q1/6874
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Quick Facts
Patent No.
US 12,319,955
App. No.
18/343,858
Granted
Jun 3, 2025
Kind
B2
Abstract

The present disclosure relates to materials and methods for spatial detection of nucleic acid in a tissue sample or a portion thereof. In particular, provided herein are materials and methods for detecting RNA so as to obtain spatial information about the localization, distribution or expression of genes in a tissue sample. In some embodiments, the materials and methods provided herein enable detection of gene expression in a single cell.

Claims (27)

1. A method of generating a spatial transcriptomics gene expression image having subcellular resolution, comprising:

a. providing a flat array surface comprising high density clusters of probes, wherein each probe comprises a spatial barcode sequence and a capture domain, and wherein a location of each cluster on said flat array surface is known;

b. contacting said flat array surface with a tissue section under conditions such that RNA from cells in said tissue section hybridizes to said capture domain of said probes;

c. reverse transcribing target sequences from said RNA on 3′ ends of said probes;

d. generating second strand copies of said probes and eluting said second strand copies from said array;

e. generating a sequencing library from eluted second strand copies;

f. sequencing said sequencing library to generate sequencing data; and

g. generating a spatial transcriptomics gene expression image having subcellular resolution from said sequencing data.

2. The method of claim 1 , wherein each probe in a given cluster comprises an identical spatial barcode sequence, and wherein said spatial barcode sequence for each cluster is unique.

3. The method of claim 1 , wherein said flat array surface comprises 0.5-2 million clusters per 1 mm 2 of surface.

4. The method of claim 3 , wherein said flat array surface comprises about 1.5 million clusters per 1 mm 2 of surface.

5. The method of claim 1 , wherein each cluster comprises at least 200 probes.

6. The method of claim 1 , wherein each cluster comprises at least 500 probes.

7. The method of claim 1 , wherein each cluster comprises at least 800 probes.

8. The method of claim 1 , wherein each cluster has a diameter of 500-1200 nm.

9. The method of claim 8 , wherein each cluster has an average diameter of 0.6 μm.

10. The method of claim 1 , wherein said flat array surface comprises a material selected from glass, silicon, poly-L-lysine coated materials, nitrocellulose, polystyrene, cyclic olefin copolymers (COCs), cyclic olefin polymers (COPs), polyacrylamide, polypropylene, polyethylene and polycarbonate.

11. The method of claim 1 , wherein said capture domain is identical for each probe.

12. The method of claim 1 , wherein said capture domain comprises a poly-T oligonucleotide comprising at least 10 deoxythymidine residues.

13. The method of claim 1 , wherein each probe further comprises a sequencing barcode.

14. The method of claim 1 , wherein each probe further comprises one or more filler sequences.

15. The method of claim 1 , wherein each probe further comprises a unique molecular identifier (UMI) barcode sequence.

16. The method of claim 1 , wherein each probe further comprises a cleavage domain comprising a binding site for a restriction endonuclease.

17. The method of claim 1 , further comprising imaging the tissue before or after reverse transcribing target sequences from said RNA on 3′ ends of said probes.

18. The method of claim 17 , further comprising correlating the identified location of each cluster on said flat array surface with a corresponding location within said tissue section.

19. The method of claim 1 , wherein the distance between centers of said clusters is 1 μm or less.

20. The method of claim 1 , wherein said clusters are produced by bridge amplification.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jul 14, 2023
From: LEE, JUN HEE
To: THE REGENTS OF THE UNIVERSITY OF MICHIGAN
Reel/Frame 064260/0511 →
Continuity (6)
Continuation 17708981 · Mar 30, 2022
Continuation PCTUS2021041725 · Jul 15, 2021
Provisional Application 63141254 · Jan 25, 2021
Provisional Application 63053238 · Jul 17, 2020
Related Publication 20240132936A1 · Apr 25, 2024
Related Publication 20240229102A9 · Jul 11, 2024
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