Enhanced utilization of surface primers in clusters

• US 10,590,464 B2
• Filed: 05/27/2016
• Issued: 03/17/2020
• Est. Priority Date: 05/29/2015
• Status: Active Grant

• Current Assignee: Illumina Cambridge Ltd. (California) (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Ltd. (California) (Illumina Incorporated)
• Inventors: Boutell, Jonathan Mark, Skinner, Gary Mark
• Primary Examiner(s): Kim, Young J
• Application Number:
  US15/576,211
• Publication Number:
  US 20180142281A1
• Time in Patent Office: 1,390 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2523/113 Denaturating agents
• CPC Class Codes: C12Q 2523/305 Denaturation or renaturation by physical action
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2565/543 characterised by the use of two or more capture oligonucleotide primers in concert, e.g. bridge amplification

Abstract

Presented herein are methods and compositions for enhancing utilization of surface primers during the surface amplification process. The methods are useful for surface amplification at improved densities. The methods and compositions provided herein enable creation of clusters which are brighter, but at the same densities as currently achieved using standard cluster amplification.

Claims

1. A method of preparing immobilized templates for a nucleic acid sequencing reaction comprising:

(a) providing a solid support having a plurality of forward and reverse amplification primers immobilized thereon, wherein a subset of said plurality of amplification primers comprises a cleavage site;

(b) amplifying a target nucleic acid template using the subset of amplification primers on the solid support to produce a plurality of double-stranded nucleic acid molecules, wherein both strands of each double-stranded nucleic acid molecule are attached to the solid support at their 5′

ends;

(c) cleaving the subset of amplification primers at the cleavage site to produce a linearized amplification product comprising a cleaved non-immobilized strand and a complementary immobilized strand; and

(d) subjecting the amplification product to partially-denaturing conditions to facilitate hybridization of a 3′

terminal portion of the cleaved non-immobilized strand with a complementary immobilized amplification primer, followed by extension of the immobilized amplification primer to generate an immobilized copy of the non-immobilized strand of the amplification product.

2. The method of claim 1, wherein the partially-denaturing conditions comprise adding one or more components of a recombinase/polymerase amplification reaction to facilitate strand invasion.

3. The method of claim 1, wherein the partially-denaturing conditions comprise subjecting the template to conditions suitable for template walking.

4. The method of claim 1, wherein step (d) comprises

applying forward or reverse amplification primers in solution to facilitate hybridization of the solution amplification primers to the non-immobilized end of the complementary immobilized strand of the amplification product.

5. The method of claim 4, wherein the subset of amplification primers on the solid support comprises forward amplification primers.

6. The method of claim 5, wherein step (d) comprises applying forward amplification primers in solution to facilitate hybridization of the solution forward amplification primers to the non-immobilized end of the complementary immobilized strand of the amplification product.

7. The method of claim 4, wherein the subset of amplification primers comprises reverse amplification primers.

8. The method of claim 7, wherein step (d) comprises applying reverse amplification primers in solution to facilitate hybridization of the solution reverse amplification primers to the non-immobilized end of the complementary immobilized strand of the amplification product.

9. The method of claim 1, further comprising sequencing the target nucleic acid.

10. The method of claim 9, wherein sequencing the target nucleic acid comprises:

hybridizing one or more sequencing primers to an immobilized nucleic acid strand;

extending the one or more sequencing primers by incorporating one or more labeled nucleotides into a nascent strand; and

detecting the labeled nucleotides, thereby obtaining sequence information about the target nucleic acid.

11. The method of claim 1, wherein said solid support is planar.

12. The method of claim 1, wherein said solid support comprises microwells.

13. The method of claim 1, wherein said target nucleic acid has a length of at least 10, 20, 50, 100, 200 or at least 500 nucleotides.

14. The method of claim 1, wherein the subset of amplification primers on the solid support are the forward amplification primers.

15. The method of claim 1, wherein the subset of amplification primers on the solid support are the reverse amplification primers.