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

• US 10,619,204 B2
• Filed: 11/11/2015
• Issued: 04/14/2020
• Est. Priority Date: 11/11/2014
• Status: Active Grant

• Current Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• 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
• Primary Examiner(s): Horlick, Kenneth R
• Application Number:
  US15/525,892
• Publication Number:
  US 20180037950A1
• Time in Patent Office: 1,616 Days
• Field of Search: None
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01L 2300/0877 Flow chambers
• CPC Class Codes: B01L 3/502707 characterised by the manufacture of the container or its components by shaping or joining plastic parts B29C59/00, B29C65/00; by laminating B32B37/00; manufacture of microstructural devices in general B81C
• 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 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2565/513 characterised by the pattern of the arrayed oligonucleotides
• CPC Class Codes: C12Q 2565/543 characterised by the use of two or more capture oligonucleotide primers in concert, e.g. bridge amplification
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 50/14 Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support

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.

Claims

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 μ

m.

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.