Kinetic exclusion amplification of nucleic acid libraries

• US 9,169,513 B2
• Filed: 10/13/2014
• Issued: 10/27/2015
• 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): Horlick, Kenneth R.
• Assistant Examiner(s): THOMAS, DAVID C
• Application Number:
  US14/512,693
• Publication Number:
  US 20150080230A1
• Time in Patent Office: 379 Days
• Field of Search: None
• US Class Current: 1/1
• 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 comprising amplicons from target nucleic acids from the solution,

wherein the plurality of different target nucleic acids is at a concentration that results in simultaneously

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

(ii) amplifying the target nucleic acids that are at the amplification sites at an amplification rate to inhibit more than one of the different target nucleic acids from being amplified at each amplification site.

2. 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.

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

4. The method of claim 3, 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.

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

6. The method of claim 3, wherein each of the features comprises a bead, well, channel, ridge, projection or combination thereof.

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

8. The method of claim 2, wherein the capture agents comprise capture nucleic acids that are complementary to the different target nucleic acids.

9. The method of claim 8, wherein the different target nucleic acids comprise universal sequences that are complementary to the capture nucleic acids.

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

11. The method of claim 10, wherein the array of amplification sites comprises an array of features on a surface.

12. The method of claim 11, 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).

13. The method of claim 1, wherein the amplifying of the target nucleic acids that are transported to the amplification sites occurs isothermally.

14. 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.

15. The method of claim 1, wherein greater than 40% of the amplification sites comprising amplicons that are produced in (b) contain a clonal population of the amplicons.

16. 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).

17. The method of claim 16, wherein the rate at which the amplicons are generated to fill the capacity of the respective amplification site exceeds the rate at which the individual target nucleic acids are transported to the individual amplification sites respectively.

18. 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.

19. The method of claim 18, where the electric field is increased as the reacting proceeds over time.

20. The method of claim 18, wherein the array of amplification sites comprises an array of non-contiguous features on a surface, the features being separated by interstitial regions of the surface.

21. The method of claim 20, wherein the different target nucleic acids are actively repelled from the interstitial regions by application of a second electric field.

22. The method of claim 21, wherein the electric field and the second electric field are simultaneously applied to the array.

23. The method of claim 21, wherein the electric field and the second electric field are applied to the array in alternate repetitions.

24. The method of claim 21, wherein the second electric field is created by application of an alternating current or a direct current to the interstitial regions.

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

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

27. The method of claim 1, further comprising

(c) performing a sequencing procedure on the array to determine the nucleotide sequences for the amplicons.

28. The method of claim 27, wherein the sequencing procedure determines nucleotide sequences for apparently clonal amplification sites.

29. The method of claim 28, wherein the apparently clonal amplification sites contain at most 5% contaminating amplicons.

30. The method of claim 6, wherein each of the features comprises a bead.

31. The method of claim 6, wherein each of the features comprises a well.

32. The method of claim 6, wherein each of the features comprises a channel.

33. The method of claim 6, wherein each of the features comprises a ridge.

34. The method of claim 6, wherein each of the features comprises a projection.

35. The method of claim 7, wherein the array of amplification sites comprises beads in solution.

36. The method of claim 7, wherein the array of amplification sites comprises beads on a surface.