Method of sequencing nucleic acid colonies formed on a surface by re-seeding

• US 9,416,415 B2
• Filed: 03/06/2015
• Issued: 08/16/2016
• Est. Priority Date: 12/23/2008
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Ronaghi, Mostafa, Eltoukhy, Helmy A.
• Primary Examiner(s): Horlick, Kenneth
• Application Number:
  US14/641,054
• Publication Number:
  US 20150211062A1
• Time in Patent Office: 529 Days
• Field of Search: 435/6.12, 435/91.2
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• 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

A method of sequencing nucleic acids, which can include steps of (a) providing a substrate having a surface having a repeating pattern of features, wherein the features are spatially separated from each other on the surface of the substrate; (b) contacting the repeating pattern of features with a solution of different target nucleic acids to seed a subset of the features that contact the solution, wherein no more than the subset of the features that contact the solution is seeded with the target nucleic acids; (c) amplifying the target nucleic acids at the subset of features; (d) repeating steps (b) and (c) to increase the number of features that are seeded with a nucleic acid, thereby making an array of different nucleic acid colonies; and (e) detecting sequencing reactions at the different nucleic acid colonies on the surface.

Claims

1. A method of sequencing nucleic acids, comprising

(a) providing a substrate comprising a surface having a repeating pattern of features, wherein the features are spatially separated from each other on the surface of the substrate;

(b) contacting the repeating pattern of features with a solution of different target nucleic acids to seed a subset of the features that contact the solution, wherein no more than the subset of the features that contact the solution is seeded with the target nucleic acids;

(c) amplifying the target nucleic acids at the subset of features;

(d) repeating steps (b) and (c) to increase the number of features that are seeded with a nucleic acid, thereby making an array of different nucleic acid colonies; and

(e) detecting sequencing reactions at the different nucleic acid colonies on the surface.

2. The method of claim 1, wherein the features comprise wells.

3. The method of claim 2, wherein the density of the wells on the surface is at least 1,000 wells/cm′

.

4. The method of claim 1, wherein the sequencing reactions comprise extension of nascent nucleic acid strands through iterative addition of nucleotides against templates in the nucleic acid colonies.

5. The method of claim 4, wherein the nucleotides comprise fluorescent labels that are detected in the sequencing reactions.

6. The method of claim 4, wherein the detecting comprises detecting a byproduct of incorporating the nucleotides into the nascent nucleic acid strands.

7. The method of claim 6, wherein the byproduct is pyrophosphate.

8. The method of claim 1, wherein the features comprise attached primer nucleic acids, and the amplifying comprises extension of the attached primer nucleic acids.

9. The method of claim 8, wherein each of the features comprises a pair of attached primer nucleic acids.

10. The method of claim 9, wherein the amplifying comprises bridge amplification.

11. The method of claim 8, wherein the amplifying comprises polymerase chain reaction amplification.

12. The method of claim 1, wherein the nucleic acid colonies are covalently attached to the features.

13. The method of claim 1, wherein the different target nucleic acids comprise a common adapter sequence.

14. The method of claim 13, wherein the seeding comprises hybridizing the common adapter sequence to a primer nucleic acid that is attached to each of the features.

15. The method of claim 1, wherein the same solution of different target nucleic acids is contacted with the substrate in steps (b) and (d).

16. The method of claim 1, wherein the substrate comprises a CMOS detector.

17. The method of claim 1, wherein the substrate comprises a density of at least 1,000 features/cm².

18. The method of claim 1, wherein each feature in the subset is seeded with no more than a single nucleic acid from the solution during step (b).

19. The method of claim 14, wherein the amplifying comprises extension of the attached primer nucleic acid.