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

• US 10,167,506 B2
• Filed: 10/02/2017
• Issued: 01/01/2019
• 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 R
• Application Number:
  US15/722,500
• Publication Number:
  US 20180044732A1
• Time in Patent Office: 456 Days
• Field of Search: None
• 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 contacting a substrate having spatially distinguishable features with a plurality of nucleic acids to seed a subset of the features, thereby generating a seeded subset; amplifying the nucleic acids in the seeded subset to form nucleic acid colonies; repeating the preceding steps to increase the number of seeded features, thereby generating an array of nucleic acid colonies; and sequencing the array of nucleic acid colonies.

Claims

1. A method of sequencing nucleic acids, the method comprising:

(a) contacting a substrate having spatially distinguishable features with a plurality of nucleic acids to seed a subset of the features, thereby generating a seeded subset;

(b) amplifying the nucleic acids in the seeded subset to form nucleic acid colonies;

(c) repeating steps (a) and (b) to increase the number of seeded features, thereby generating an array of nucleic acid colonies; and

(d) sequencing the array of nucleic acid colonies.

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

3. The method of claim 2, wherein the density of the wells is at least 1,000 wells/cm².

4. The method of claim 1, wherein the sequencing comprises 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 sequencing 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 a polymerase chain reaction.

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 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 plurality of nucleic acids is contacted with the substrate in steps (a) and (c).

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 14, wherein the amplifying comprises extension of the attached primer nucleic acid.