Kinetic exclusion amplification of nucleic acid libraries

• US 10,385,384 B2
• Filed: 08/08/2017
• Issued: 08/20/2019
• 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 L., Venkatesan, Bala Murali, Bowen, M. Shane, Vijayan, Kandaswamy
• Primary Examiner(s): Thomas, David C
• Application Number:
  US15/672,161
• Publication Number:
  US 20170335380A1
• Time in Patent Office: 742 Days
• 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 amplifying nucleic acids, comprising

(a) providing an amplification reagent comprising

(i) an array 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 amplification sites in the array,

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 to produce a plurality of amplification sites that each comprise a clonal population of amplicons from an individual target nucleic acid from the solution,

wherein the reacting comprises (i) producing a first amplicon from an individual target nucleic acid that transports to each of the amplification sites, and (ii) producing subsequent amplicons from the individual target nucleic acid that transports to each of the amplification sites or from the first amplicon, wherein the average rate at which the subsequent amplicons are generated at the amplification sites exceeds the average rate at which the first amplicon is generated at the amplification sites.

2. The method of claim 1, wherein each of the amplification sites comprises a plurality of primers that are used to produce the amplicons in (b).

3. The method of claim 2, wherein the producing of the first amplicon comprises converting at least one of the primers from a non-extendible state to an extendible state.

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

5. The method of claim 4, wherein the features are non-contiguous and are separated by interstitial regions of the surface that lack the primers that are used to produce the amplicons in (b).

6. The method of claim 1, wherein the reacting comprises transporting target nucleic acids from the solution to the amplification sites simultaneously with (i) the producing of the first amplicon, and (ii) the producing of the subsequent amplicons.

7. The method of claim 1, wherein the target nucleic acids are transported from the solution to the amplification sites prior to (i) the producing of the first amplicon, and (ii) the producing of the subsequent amplicons.

8. The method of claim 1, wherein each of the amplification sites comprises a plurality of capture agents capable of binding to the different target nucleic acids in the solution.

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

10. The method of claim 9, wherein the area for each of the features is greater than the average diameter of the excluded volume of the individual target nucleic acids that are transported to each of the amplification sites.

11. The method of claim 10, wherein the features are non-contiguous and are separated by interstitial regions of the surface that lack the capture agents.

12. The method of claim 9, wherein each of the features comprises a bead, well, channel, ridge, projection or combination thereof.

13. The method of claim 8, wherein the capture agents comprise capture nucleic acids that are complementary to the different target nucleic acids.

14. The method of claim 13, wherein the different target nucleic acids comprise universal sequences that are complementary to the capture nucleic acids.

15. The method of claim 8, wherein the capture agent comprises a receptor that binds to a ligand attached to the different target nucleic acids.

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

17. The method of claim 16, wherein the amplification reagent further comprises a recombinase and a single stranded binding protein.

18. The method of claim 17, wherein the amplification reagent further comprises a molecular crowding agent.

19. The method of claim 1, wherein the reacting occurs isothermally.

20. The method of claim 1, wherein the reacting does not include a denaturation cycle.

21. The method of claim 1, wherein the reacting does not include replacing the solution with a chemical reagent that denatures the target nucleic acids and the amplicons during the reacting.

22. The method of claim 1, wherein the reacting does not include heating the solution to a temperature that denatures the target nucleic acids and the amplicons during the reacting.

23. The method of claim 1, wherein the different target nucleic acids are double stranded DNA molecules.

24. The method of claim 1, wherein the different target nucleic acids have an average strand length that is less than 1,000 nucleotides.

25. The method of claim 1, wherein the plurality of amplification sites that comprise a clonal population of amplicons exceeds 40% of the amplification sites in the array.

26. 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).

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