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

Patent metadata

Field	Value
Publication number	US-9416415-B2
Application number	US-201514641054-A
Country	US
Kind code	B2
Filing date	Mar 6, 2015
Priority date	Dec 23, 2008
Publication date	Aug 16, 2016
Grant date	Aug 16, 2016

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.

First claim

What is claimed is:

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/cm 2 .

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.

Assignees

• Illumina Inc

Inventors

• Ronaghi Mostafa
• Eltoukhy Helmy A

Classifications

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Q1/6837

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

• C12Q1/6874 Primary

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

• C12Q2535/122

  Massive parallel sequencing · CPC title

• C12Q2565/543

  characterised by the use of two or more capture oligonucleotide primers in concert, e.g. bridge amplification · CPC title