Capturing and amplifying polynucleotides, and reusing flowcells, using silica particles

Patent metadata

Field	Value
Publication number	US-2024417792-A1
Application number	US-202418736835-A
Country	US
Kind code	A1
Filing date	Jun 7, 2024
Priority date	Jun 8, 2023
Publication date	Dec 19, 2024
Grant date	—

Abstract

In some examples, a method of sequencing a polynucleotide includes hybridizing the polynucleotide to a first oligonucleotide coupled to a particle. The hybridized polynucleotide may be amplified using additional oligonucleotides coupled to the particle, to generate amplicons coupled to the particle. After the amplifying, the particle, having the amplicons coupled thereto, may be disposed within a flowcell. After disposing the particle within the flowcell, the particle may be dissolved, leaving the amplicons within the flowcell. The flowcell may be used to sequence the amplicons.

First claim

1 . A method of sequencing a polynucleotide, the method comprising:
hybridizing the polynucleotide to a first oligonucleotide coupled to a particle; amplifying the hybridized polynucleotide using additional oligonucleotides coupled to the particle, to generate amplicons coupled to the particle; after the amplifying, disposing the particle, having the amplicons coupled thereto, within a flowcell; after disposing the particle within the flowcell, dissolving the particle, leaving the amplicons within the flowcell; and using the flowcell to sequence the amplicons.

2 . The method of claim 1 , wherein disposing the particle, having the amplicons coupled thereto, within the flowcell comprises electrostatically attracting the particle or the amplicons to a region of the flowcell.

3 . The method of claim 1 , wherein the region of the flowcell comprises a recess.

4 . The method of claim 3 , wherein the recess comprises a hydrogel.

5 . The method of claim 3 , wherein the recess comprises a plurality of positively charged moieties.

6 . The method of claim 1 , wherein the particle is dissolved while using the flowcell to sequence the amplicons.

7 . The method of claim 1 , wherein the particle is dissolved before using the flowcell to sequence the amplicons.

8 . The method of claim 1 , wherein the oligonucleotides are coupled to a polymer that is coupled to the particle.

9 . The method of claim 8 , wherein dissolving the particle further leaves the polymer within the flowcell, the amplicons being coupled to the polymer left within the flowcell.

10 . The method of claim 9 , further comprising, after sequencing the amplicons, removing the polymer and the amplicons.

11 . The method of claim 10 , wherein the polymer and the amplicons are removed using a basic solution.

12 . The method of claim 8 , wherein the polymer is coupled to the particle via a siloxane (—Si—O—Si—) bond.

13 . The method of claim 8 , wherein the polymer comprises polyacrylamide.

14 . The method of claim 8 , wherein before the particle is dissolved, the polymer is covalently coupled to the particle.

15 . The method of claim 8 , wherein both before and after the particle is dissolved, the polymer is covalently coupled to the amplicons.

16 . The method of claim 8 , wherein both before and after the particle is dissolved, the polymer is non-covalently coupled to the flowcell.

17 . The method of claim 1 , wherein the particle comprises silica.

18 . The method of claim 1 , wherein the particle is dissolved using a basic solution.

19 . (canceled)

20 . A method of sequencing a polynucleotide using a particle having a plurality of oligonucleotides coupled thereto, the method comprising:
using the plurality of oligonucleotides to generate amplicons of the polynucleotide that are coupled to the particle; disposing the particle, having the amplicons coupled thereto, within a flowcell; after disposing the particle within the flowcell, dissolving the particle; and using the flowcell to sequence the amplicons.

21 . A method of using a flowcell, the method comprising:
disposing a first particle, having a first set of polynucleotide amplicons coupled thereto, within the flowcell; dissolving the first particle, leaving the first set of polynucleotide amplicons within the flowcell; using the flowcell to sequence the first set of polynucleotide amplicons; after sequencing the first set of polynucleotide amplicons, removing the first set of polynucleotide amplicons from the flowcell; and after removing the first set of polynucleotide amplicons from the flowcell, disposing a second particle, having a second set of polynucleotide amplicons coupled thereto, within the flowcell.

22 - 50 . (canceled)

Assignees

• Illumina Inc

Inventors

• La Rosa Angelo
• Von Hatten Xavier

Classifications

• C12Q1/6844

  Nucleic acid amplification reactions · CPC title

• C12Q1/6834

  Enzymatic or biochemical coupling of nucleic acids to a solid phase · CPC title

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q1/6874 Primary

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

• C12Q1/6869 Primary

  Methods for sequencing · CPC title