Compositions and methods for nucleic acid sequencing

Patent metadata

Field	Value
Publication number	US-12460261-B2
Application number	US-202418988274-A
Country	US
Kind code	B2
Filing date	Dec 19, 2024
Priority date	Feb 6, 2019
Publication date	Nov 4, 2025
Grant date	Nov 4, 2025

Abstract

Provided herein are methods and compositions for improved sequencing techniques using, for example, polymeric particles and/or three-dimensional structures.

First claim

What is claimed is:

1. A method of detecting polynucleotides in a polymer scaffold, the method comprising:
(a) amplifying polynucleotides to produce discrete amplicon clusters within a three-dimensional polymer scaffold, wherein the amplicons are covalently bound to the polymer scaffold;
(b) contacting the polymer scaffold with a plurality of fluorescently labeled molecules and, within two or more discrete amplicon clusters, binding a fluorescently labeled molecule to a nucleic acid molecule in the amplicon cluster;
(c) detecting the fluorescently labeled molecules to identify the polynucleotide in the polymer scaffold.

2. The method of claim 1 , wherein detecting the fluorescently labeled molecules further identifies three-dimensional coordinates of the polynucleotide.

3. The method of claim 2 , further comprising forming an image based on the detected labels.

4. The method of claim 1 , prior to (a), comprising contacting the polymer scaffold with a plurality of primers and covalently attaching the primers to the polymer scaffold.

5. The method of claim 4 , wherein the plurality comprises a first primer comprising a first capture sequence and a second primer comprising a second capture sequence.

6. The method of claim 5 , comprising binding a first template polynucleotide to the first capture sequence, wherein the first template polynucleotide comprises a first primer binding sequence.

7. The method of claim 1 , comprising cross-linking the amplicons to the polymer scaffold.

8. The method of claim 1 , wherein the three-dimensional polymer scaffold is in the channel of a flow cell.

9. The method of claim 1 , wherein amplifying comprises isothermal amplification.

10. The method of claim 1 , wherein amplifying comprises rolling circle amplification.

11. The method of claim 1 , wherein the fluorescently labeled molecules are fluorescently labeled nucleotides.

12. The method of claim 11 , wherein each fluorescently labeled nucleotide comprises a fluorophore moiety covalently attached to a nucleotide molecule via a cleavable linker.

13. The method of claim 12 , further comprising cleaving the cleavable linker and removing the fluorophore moiety and repeating (b) and (c).

14. The method of claim 13 , wherein detecting comprises imaging multiple two-dimensional planes.

15. The method of claim 1 , wherein the fluorescently labeled molecules are fluorescently labeled oligonucleotides.

16. The method of claim 15 , further comprising removing the fluorescently labeled oligonucleotides and repeating (b) and (c).

17. The method of claim 16 , wherein detecting comprises imaging multiple two-dimensional planes.

18. The method of claim 1 , wherein the polynucleotides comprise a gene sequence or a portion thereof.

19. The method of claim 1 , wherein the polymer scaffold comprises polyacrylamide.

20. The method of claim 1 , wherein the polymer scaffold comprises polyacrylate.

Assignees

• Singular Genomics Systems Inc

Inventors

• Glezer Eli N
• Varnosfaderani Mohammad Vatankhah
• Witters Daan
• Karimkhani Vahid

Classifications

• G01N33/544

  the carrier being organic · CPC title

• G01N33/5308

  for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites · CPC title

• C12Q1/6874 Primary

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q1/6844

  Nucleic acid amplification reactions · CPC title

• C12Q2565/601

  being a microscope, e.g. atomic force microscopy [AFM] · CPC title