Kinetic exclusion amplification of nucleic acid libraries

• US 9,758,816 B2
• Filed: 10/09/2015
• Issued: 09/12/2017
• Est. Priority Date: 06/15/2012
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Shen, Min-Jui Richard, Boutell, Jonathan Mark, Stephens, Kathryn M., Ronaghi, Mostafa, Gunderson, Kevin, Venkatesan, Bala Murali, Bowen, M. Shane, Vijayan, Kandaswamy
• Primary Examiner(s): THOMAS, DAVID C
• Application Number:
  US14/879,932
• Publication Number:
  US 20160053310A1
• Time in Patent Office: 704 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 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00587 High throughput processes
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/00621 by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00653 the compounds being bound to electrodes embedded in or on the solid supports
• CPC Class Codes: B01J 2219/00675 In-situ synthesis on the substrate
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/507 Recombinase
• CPC Class Codes: C12Q 2527/101 Temperature
• CPC Class Codes: C12Q 2527/113 Time
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2565/513 characterised by the pattern of the arrayed oligonucleotides
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer

Abstract

A method including (a) providing an amplification reagent including an array of sites, and a solution having different target nucleic acids; and (b) reacting the amplification reagent to produce amplification sites each having a clonal population of amplicons from a target nucleic acid from the solution. The reacting can include simultaneously transporting the nucleic acids to the sites at an average transport rate, and amplifying the nucleic acids that transport to the sites at an average amplification rate, wherein the average amplification rate exceeds the average transport rate. The reacting can include producing a first amplicon from a nucleic acid that transports to each of the sites, and producing subsequent amplicons from the nucleic acid or from the first amplicon, wherein the average rate at which the subsequent amplicons are generated exceeds the average rate at which the first amplicon is generated.

Claims

1. A method for determining nucleic acid sequences, comprising performing a sequencing procedure that detects an apparently clonal population of amplicons at each of a plurality of amplicon sites on an array, wherein the array is made by a process that comprises

(a) providing an amplification reagent comprising

(i) a plurality of amplification sites, and

(ii) a solution comprising a plurality of different target nucleic acids,

wherein the number of the different target nucleic acids in the solution exceeds the number of the amplification sites,

wherein the different target nucleic acids have fluidic access to the plurality of amplification sites, and

wherein each of the amplification sites comprises a capacity for several nucleic acids in the plurality of different nucleic acids;

and

(b) reacting the amplification reagent, wherein the plurality of different target nucleic acids in the solution is at a concentration that results in simultaneously;

(i) transporting the different target nucleic acids from the solution to the amplification sites, and

(ii) amplifying the target nucleic acids that are at the amplification sites at an amplification rate that promotes filling the capacity of the amplification sites and exclusion of more than one of the different target nucleic acids from attaching at each amplification site to produce an array of amplicon sites that each comprise the apparently clonal population of amplicons.

2. The method of claim 1, wherein the array of amplification sites comprises an array of features on a substrate.

3. The method of claim 2, wherein the area for each of the features is greater than the diameter of the excluded volume of the target nucleic acids that are transported to the amplification sites.

4. The method of claim 3, wherein the features are non-contiguous and are separated by interstitial regions that lack the capture agents.

5. The method of claim 2, wherein each of the features comprises a well.

6. The method of claim 5, wherein the wells do not contain beads.

7. The method of claim 1, wherein the plurality of amplification sites comprises beads in solution or beads on a substrate.

8. The method of claim 1, wherein each of the amplification sites is attached to a plurality of capture nucleic acids that are complementary to universal sequences in the different target nucleic acids.

9. The method of claim 1, wherein each of the amplification sites is attached to a plurality of primers that are used to amplify the target nucleic acids in (b).

10. The method of claim 1, wherein the amplifying of the target nucleic acids that are transported to the amplification sites does not include a denaturation cycle.

11. The method of claim 1, wherein a sufficient number of amplicons are generated from the individual target nucleic acids at the individual amplification sites respectively to fill the capacity of the respective amplification site during (b).

12. The method of claim 1, wherein the different target nucleic acids are actively transported to the amplification sites assisted by application of an electric field.

13. The method of claim 1, wherein the transporting comprises passive diffusion.

14. The method of claim 1, wherein the amplification reagent further comprises a polymerase and a recombinase.

15. The method of claim 1, wherein the apparently clonal amplification sites contain at most 5% contaminating amplicons.

16. The method of claim 1, wherein the sequencing procedure comprises using the amplicons as templates in a sequencing-by-synthesis procedure.

17. The method of claim 1, wherein the sequencing procedure comprises repeated delivery of reagents to the array of amplicon sites in multiple cycles.

18. The method of claim 1, wherein the sequencing procedure comprises detection of labeled nucleotides incorporated into an extension product on the array of amplicon sites.

19. The method of claim 18, wherein the labeled nucleotides comprise fluorescent labels.

20. The method of claim 1, wherein the sequencing procedure comprises detection of protons released upon incorporation of nucleotides into an extension product on the array of amplicons.

21. The method of claim 1, wherein each of the amplicon sites occupies an area that is less than 100 μ

m² on the array.

22. The method of claim 1, wherein the sequencing procedure and the process of making the array are carried out on an integrated device, wherein the integrated device comprises a detector that detects the amplicon sites in the sequencing procedure, and fluidic components that deliver fluids of the amplification reagent to the plurality of amplification sites.

23. The method of claim 22, wherein the integrated device comprises fluidic components that deliver fluids of the sequencing procedure to the array of amplicon sites.

24. A nucleic acid sequencing system, comprising:

a system controller that is programmed to direct the performance of the method of claim 1;

a detector that is configured to detect the amplicon sites; and

fluidic components that are configured to deliver fluids of the amplification reagent to the plurality of amplification sites.

25. The system of claim 24, further comprising fluidic components that are configured to deliver fluids of the sequencing procedure to the array of amplicon sites.