Methods and arrays for producing and sequencing monoclonal clusters of nucleic acid

Patent metadata

Field	Value
Publication number	US-10619204-B2
Application number	US-201515525892-A
Country	US
Kind code	B2
Filing date	Nov 11, 2015
Priority date	Nov 11, 2014
Publication date	Apr 14, 2020
Grant date	Apr 14, 2020

Abstract

Embodiments of the present disclosure relate to methods for capturing and amplifying target polynucleotides on a solid surface, in particular in a well in a microarray, wherein the microarray may comprise a) a substrate comprising at least one well, a surface surrounding the well and an inner well surface; b) a first layer covering the inner well surface and comprising at least one first capture primer pair; and c) a second layer covering the first layer and the surface surrounding the well.

First claim

What is claimed is:

1. A microarray comprising:
a) a substrate comprising at least one well, a surface surrounding the well and an inner well surface;
b) a first layer covering the inner well surface and comprising at least one first capture primer pair; and
c) a second layer covering the first layer and the surface surrounding the well.

2. The microarray of claim 1 , wherein the first layer does not cover the surface surrounding the well.

3. The microarray of claim 1 , wherein the first layer at least partially covers the inner well surface.

4. The microarray of claim 1 , wherein the at least one well is a plurality of wells.

5. The microarray of claim 4 , wherein the plurality of wells are spaced at a pitch of about 700 nm.

6. The microarray of claim 1 , wherein the diameter of the well is less than, or about 1 μm.

7. The microarray of claim 6 , wherein the diameter of the well is between about 100 nm and 400 nm.

8. The microarray of claim 1 , wherein the first layer comprises a first polymer coating, and wherein the second layer optionally comprises a second polymer coating.

9. The microarray of claim 8 , wherein the first polymer coating comprises poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide (PAZAM), and wherein the second polymer coating comprises PAZAM or silane free acrylamide (SFA).

10. The microarray claim 1 , wherein the at least one first capture primer pair is a plurality of first capture primer pairs, and wherein primers of the at least one first capture primer pair optionally comprise a universal capture region.

11. The microarray of claim 10 , wherein the first primer of the at least one first capture primer pair comprises a primer nucleotide sequence of SEQ ID NO. 1 and the second primer of the at least one first capture primer pair comprises a primer nucleotide sequence of SEQ ID NO. 2.

12. The microarray of claim 11 , wherein the primers of the at least one first capture primer pair further comprises a sequencing primer binding site (SBS).

13. The microarray of claim 12 , wherein the first primer of the at least one first capture primer pairs comprises the primer nucleotide sequence of SEQ ID NO. 1 and a primer nucleotide sequence of SEQ ID NO. 5, and the second primer of the at least one first capture primer pair comprises the primer nucleotide sequence of SEQ ID NO. 2 and a primer nucleotide sequence of SEQ ID NO. 6.

14. The microarray of claim 1 , wherein the second layer comprises at least one second capture primer pair, and wherein the at least one second capture primer pair is optionally a plurality of second capture primer pairs.

15. The microarray of claim 14 , wherein the primers of the at least one second capture primer pair are blocked at the 3′-end.

16. The microarray of claim 15 , wherein the primers of the at least one second capture primer pair are 3′-phosphate-terminated, and wherein the 3′-phosphate terminated primers of the at least one second capture primer pair optionally comprise a universal capture region.

17. The microarray of claim 16 , wherein the first primer of the at least one second capture primer pair comprises the primer nucleotide sequence of SEQ ID NO. 1 and the second primer of the at least one second capture primer pair comprises the primer nucleotide sequence of SEQ ID NO. 2.

18. The microarray of claim 14 , wherein the primers of the at least one second capture primer pair are not blocked at the 3′-end, and wherein the primers of the at least one second capture primer pair optionally comprise a universal capture region.

19. The microarray of claim 18 , wherein the first primer of the at least one second capture primer pair comprises the primer nucleotide sequence of SEQ ID NO. 1 and the second primer of the at least one second capture primer pair comprises the primer nucleotide sequence of SEQ ID NO. 2.

20. The microarray of claim 1 , wherein the at least one first capture primer pair is a plurality of first capture primer pairs, and wherein a plurality of capture primers of the plurality of first capture primer pairs each are attached to a target polynucleotide.

21. The microarray of claim 20 , wherein the plurality of target polynucleotides form a monoclonal population of target polynucleotides in the at least one well.

22. The microarray of claim 21 , wherein the at least one well comprises a plurality of wells and wherein two or more wells of the plurality of wells comprise a monoclonal population of target polynucleotides.

23. The microarray of claim 22 , wherein the two or more wells of the plurality of wells comprise a monoclonal population of the same target polynucleotide or of two or more different target polynucleotides.

24. The microarray of claim 14 , wherein the at least one first capture primer pair is a plurality of first capture primer pairs and the at least one second capture primer pair is a plurality of second capture primer pairs, and wherein a plurality of primers of the plurality of first capture primer pairs and the plurality of second capture primer pairs is attached to a plurality of target polynucleotides.

25. A method for amplifying a nucleic acid, comprising:
a) producing a first layer on a substrate, wherein the substrate comprises at least one well, a surface surrounding the well and an inner well surface, wherein the first layer covers the inner well surface;
b) depositing at least one first capture primer pair in the first layer;
c) producing a second layer on the substrate covering the first layer and the surface surrounding the well;
d) contacting a sample comprising a plurality of target polynucleotides with the substrate under conditions sufficient for a target polynucleotide to hybridize with a capture primer of the at least one first capture primer pair, and
e) performing a first kinetic exclusion assay (KEA) to produce a clonal population of amplicons from the target polynucleotide inside the well, thereby amplifying the target polynucleotide.

Assignees

• Illumina Cambridge Ltd

Inventors

• Gunderson Kevin L
• Bai Jingwei
• Kellinger Matthew William
• Beierle John M
• Boutell Jonathan Mark
• Rigatti Roberto
• Rogert Bacigalupo Maria Candelaria
• Boyanov Boyan
• Maisinger Klaus

Classifications

• C12Q1/6837 Primary

  using probe arrays or probe chips (C12Q1/6874 takes precedence) · CPC title

• B01J2219/00596

  Solid-phase processes · CPC title

• C12Q1/6874 Primary

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

• C12Q1/6853

  using modified primers or templates · CPC title

• B01J2219/00608

  DNA chips · CPC title