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EP-4735624-A1 - CAPTURING AND AMPLIFYING POLYNUCLEOTIDES USING PARTICLES

EP4735624A1EP 4735624 A1EP4735624 A1EP 4735624A1EP-4735624-A1

Abstract

In some examples, a method for capturing and amplifying a polynucleotide includes capturing the polynucleotide at a particle comprising a first region and a second region. The first region may include a first moiety that captures the polynucleotide. The second region may include a plurality of amplification primers and have a surface area which is substantially larger than the surface area of the first portion. The method includes using the plurality of amplification primers to amplify the captured polynucleotide.

Inventors

  • HONG, Sahngki
  • SHEN, FEI
  • KRAFT, LEWIS

Assignees

  • Illumina, Inc.

Dates

Publication Date
20260506
Application Date
20240625

Claims (20)

  1. 1. A method for capturing and amplifying a polynucleotide, the method comprising: capturing the polynucleotide at a particle comprising a first region and a second region, the first region comprising a first moiety that captures the polynucleotide. the second region comprising a plurality of amplification primers and having a surface area which is substantially larger than the surface area of the first portion; and using the plurality of amplification primers to amplify the captured polynucleotide.
  2. 2. The method of claim 1 , wherein the captured polynucleotide inhibits capture of any other polynucleotide at the first region of the particle.
  3. 3. The method of claim 1 or claim 2, wherein the surface area of the first portion inhibits capture of more than one polynucleotide.
  4. 4. The method of any one of claims 1 to 3, wherein the first region has a dimension which is approximately equal to or smaller than a diameter of gyration of the polynucleotide.
  5. 5. The method of any one of claims 1 to 4, wfierein the surface area of the first region is approximately 20 nm 2 or less.
  6. 6. The method of any one of claims 1 to 5, wherein the first region forms less than about 10% of an overall surface area of the particle.
  7. 7. The method of any one of claims 1 to 6, wherein the first region is formed using a first material, and the second region is formed using a second material that is different than the first material.
  8. 8. The method of claim 7, wherein at least one of the first and second regions comprises a hydrogel.
  9. 9. The method of any one of claims 1 to 8, wherein the first moiety comprises a capture primer having a sequence that is orthogonal to sequences of the amplification primers.
  10. 10. The method of claim 9, wherein the polynucleotide comprises a single-stranded primer that is substantially complementary to, and hybridizes to, the capture primer.
  11. 11. The method of claim 7 or claim 10, wherein the polynucleotide comprises at least one adapter which is complementary to an amplification primer of the plurality of amplification primers.
  12. 12. The method of any one of claims 1 to 11, wherein the first moiety covalently or non- covalently bonds to a second moiety coupled to the polynucleotide.
  13. 13. The method of any one of claims 1 to 12, wherein the plurality of amplification primers comprises a first type of amplification primers and a second type of amplification primers having a sequence that is orthogonal to a sequence of the first type of amplification primers.
  14. 14. The method of claim 13, wherein the first type of amplification primers comprises an excision moiety.
  15. 15. The method of any one of claims 1 to 14, wherein the captured polynucleotide is double stranded.
  16. 16. The method of claim 15, wherein the captured polynucleotide is amplified using strand invasion.
  17. 17. The method of any one of claims 1 to 14, wherein the captured polynucleotide is single stranded.
  18. 18. The method of claim 17, wherein the captured polynucleotide is amplified using bridge amplification.
  19. 19. The method of claim 17 or claim 18, wherein the particle further comprises oligonucleotides which are hybridized to respective amplification primers when the polynucleotide is captured.
  20. 20. The method of any one of claims 1 to 19, further comprising contacting the particle with a substrate.

Description

CAPTURING AND AMPLIFYING POLYNUCLEOTIDES USING PARTICLES CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Patent Application No. 63/510,875, filed June 28, 2023 and entitled “Capturing and Amplifying Polynucleotides Using Particles,’' the entire contents of which are incorporated by reference herein. INCORPORATION BY REFERENCE OF SEQUENCE LISTING [0002] The material in the accompanying sequence listing is hereby incorporated by reference into the application. The accompanying sequence listing XML file, named “IP- 2435-PCT.xml”, was created on June 20, 2024 and is 16 kB in size. FIELD [0003] This application generally relates to capturing and amplifying polynucleotides. BACKGROUND [0004] Cluster amplification is an approach to amplifying polynucleotides, for example for use in genetic sequencing. Target polynucleotides are captured by primers (e.g., P5 and P7 primers) coupled to a substrate surface in a flowcell, and form “seeds” at random locations on the surface. Cycles of amplification (e.g., bridge amplification) are performed to form clusters on the surface around each seed. The clusters include copies, and complementary copies, of the seed polynucleotides. In some circumstances, the substrate is patterned so as to define regions that bound different clusters, such as wells that may be filled with respective clusters. SUMMARY [0005] Examples provided herein are related to capturing and amplifying polynucleotides using particles. Particles for performing such capture and amplification, and methods of making such particles, also are disclosed. [0006] Some examples herein provide a method for capturing and amplifying a polynucleotide. The method may include capturing the polynucleotide at a particle including a first region and a second region. The first region includes a first moiety that captures the polynucleotide. The second region includes a plurality of amplification primers and has a surface area which is substantially larger than the surface area of the first portion. The method includes using the plurality of amplification primers to ampli fy the captured polynucleotide. [0007] In some examples, the captured polynucleotide inhibits capture of any other polynucleotide at the first region of the particle. In some examples, the surface area of the first portion inhibits capture of more than one polynucleotide. [0008] In some examples, the first region has a dimension which is approximately equal to or smaller than a diameter of gyration of the polynucleotide. In some examples, the surface area of the first region is approximately 20 nm2 or less. In some examples, the first region forms less than about 10% of an overall surface area of the particle. [0009] In some examples, the first region is formed using a first material, and the second region is formed using a second material that is different than the first material. In some examples, at least one of the first and second regions includes a hydrogel. [0010] In some examples, the first moiety includes a capture primer having a sequence that is orthogonal to sequences of the amplification primers. In some examples, the polynucleotide includes a single-stranded primer that is substantially complementary to, and hybridizes to, the capture primer. In some examples, the polynucleotide includes at least one adapter which is complementary to an amplification primer of the plurality of amplification primers. [0011] In some examples, the first moiety covalently or non-covalently bonds to a second moiety coupled to the polynucleotide. [0012] In some examples, the plurality of amplification primers includes a first type of amplification primers and a second type of amplification primers having a sequence that is orthogonal to a sequence of the first type of amplification primers. In some examples, the first type of amplification primers includes an excision moiety. [0013] In some examples, the captured polynucleotide is double stranded. In some examples, the captured polynucleotide is amplified using strand invasion. [0014] In some examples, the captured polynucleotide is single stranded. In some examples, the captured polynucleotide is amplified using bridge amplification. In some examples, the particle further includes oligonucleotides which are hybridized to respective amplification primers when the polynucleotide is captured. [0015] In some examples, the method further includes contacting the particle with a substrate. In some examples, the method further includes electrostatically attracting the particle to the substrate. In some examples, the particle is contacted with the substrate after capturing the polynucleotide with the particle. In some examples, the particle is contacted with the substrate after amplifying the captured polynucleotide. [0016] Some examples herein provide a method of sequencing a polynucleotide. The method may include capturing and amplifying the polynucleotide using any one