Combinatorial decoding of random nucleic acid arrays

• US 8,206,917 B2
• Filed: 06/13/2011
• Issued: 06/26/2012
• Est. Priority Date: 05/20/1999
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Chee, Mark S., Walt, David R.
• Primary Examiner(s): Kim, Young J
• Application Number:
  US13/159,302
• Publication Number:
  US 20110251101A1
• Time in Patent Office: 379 Days
• Field of Search: None
• US Class Current: 435/6.1
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00572 Chemical means
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00648 by the use of solid beads
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00702 Processes involving means for analysing and characterising the products
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2563/155 Particles of a defined size, e.g. nanoparticles
• CPC Class Codes: C12Q 2563/161 Vesicles, e.g. liposome
• CPC Class Codes: C12Q 2563/185 Nucleic acid dedicated to use as a hidden marker/bar code, e.g. inclusion of nucleic acids to mark art objects or animals
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array
• CPC Class Codes: C40B 20/04 Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding processes
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 70/00 Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or bar codes

Abstract

Methods disclosed herein relate to identification of nucleotides in a nucleotide sequence.

Claims

1. A method of identifying a nucleotide in a candidate sequence, said method comprising the steps of:

a) providing an array substrate comprising a surface having a first subpopulation and a second subpopulation of identifier probes distributed thereon, wherein identifier probes of the first subpopulation and identifier probes of the second subpopulation comprise a primer sequence adjacent to a candidate sequence, the primer sequence of the first subpopulation of identifier probes having the same sequence as the primer sequence of the second subpopulation of identifier probes and the candidate sequence of the first subpopulation of identifier probes being different from the candidate sequence of the second subpopulation of identifier probes, wherein identifier probes of the first subpopulation and identifier probes of the second subpopulation are hybridized to a plurality of detection probes comprising a variable region, said variable region comprising at least one detection nucleotide at a detection position, said detection nucleotide being able to basepair with a nucleotide of at least one of said different candidate sequences, wherein said plurality of detection probes comprises different groups, and wherein detection probes from different groups comprise a different detection nucleotide at the same detection position and a label that distinguishes the detection nucleotide;

b) detecting the presence of said label;

c) dehybridizing said detection probes from said identifier probes; and

d) associating the label with the identity of a nucleotide in the candidate sequence.

2. The method of claim 1, wherein said first and second subpopulations of identifier probes are attached to beads.

3. The method of claim 2, wherein said beads are randomly distributed on the surface of the substrate.

4. The method of claim 1, wherein said candidate sequence comprises genomic DNA.

5. The method of claim 1, wherein said candidate sequence comprises a copy of a genomic DNA fragment.

6. The method of claim 1, wherein said identifier probes are prepared by an amplification technique.

7. The method of claim 1, wherein said plurality of detection probes comprises at least four groups.

8. The method of claim 1, wherein said variable region comprises two detection nucleotides.

9. The method of claim 8, wherein the two detection nucleotides are internal nucleotides of the variable region.

10. The method of claim 8, wherein the first of the two detection nucleotides is at an end of the variable region.

11. The method of claim 10, wherein the second of the two detection nucleotides is immediately adjacent to the first detection nucleotide.

12. The method of claim 11, wherein said variable region further comprises a plurality of random nucleotides immediately adjacent to the second detection nucleotide.

13. The method of claim 12, wherein said plurality of random nucleotides consists of three random nucleotides.

14. The method of claim 13, wherein said variable region further comprises a plurality of universal bases immediately adjacent to the plurality of random nucleotides.

15. The method of claim 14, wherein said plurality of universal bases consists of three universal bases.

16. The method of claim 1, wherein said variable region comprises a single detection nucleotide.

17. The method of claim 16, wherein the single detection nucleotide is an internal nucleotide of the variable region.

18. The method of claim 16, wherein the single detection nucleotide is at an end of the variable region.

19. The method of claim 18, wherein said variable region further comprises a plurality of random nucleotides immediately adjacent to the single detection nucleotide.

20. The method of claim 19, wherein said plurality of random nucleotides consists of at least two but not more than five random nucleotides.

21. The method of claim 20, wherein said variable region further comprises a plurality of universal bases immediately adjacent to the plurality of random nucleotides.

22. The method of claim 21, wherein said plurality of universal bases consists of at least two but not more than five universal bases.

23. The method of claim 1, wherein said label is located at one end of said variable region.

24. The method of claim 1, wherein said label is located at the detection position.

25. The method of claim 1, wherein the candidate sequence comprises no more than 1000 nucleotides.

26. The method of claim 25 further comprising detecting a sufficient number of labels to permit identification of at least 20 consecutive nucleotides of the candidate sequence prior to associating said labels with the identity of a nucleotide in the candidate sequence.

27. The method of claim 1, wherein said plurality of detection probes further comprises a priming sequence complementary to a portion of the primer sequence.

28. The method of claim 27, wherein said variable region comprises two detection nucleotides.

29. The method of claim 28, wherein the first of the two detection nucleotides is immediately adjacent to said priming sequence.

30. The method of claim 29, wherein the second of the two detection nucleotides is immediately adjacent to the first detection nucleotide.

31. The method of claim 27, wherein said variable region comprises a single detection nucleotide.

32. The method of claim 31, wherein the single detection nucleotide is immediately adjacent to said priming sequence.

33. A method of identifying a nucleotide in a candidate sequence, said method comprising the steps of:

a) providing an array substrate comprising a surface having a first subpopulation and a second subpopulation of identifier probes distributed thereon, wherein identifier probes of the first subpopulation and identifier probes of the second subpopulation comprise a primer sequence adjacent to a candidate sequence, the primer sequence of the first subpopulation of identifier probes having the same sequence as the primer sequence of the second subpopulation of identifier probes and the candidate sequence of the first subpopulation of identifier probes being different from the candidate sequence of the second subpopulation of identifier probes, wherein an identifier probe of the first subpopulation is hybridized to a first detection probe and an identifier probe of the second subpopulation is hybridized to a second detection probe, each detection probe comprising a variable region, each variable region comprising at least one detection nucleotide that will basepair with a nucleotide of at least one of said different candidate sequences, wherein the first detection probe and the second detection probe have a different detection nucleotide at the same position and a label that distinguishes the detection nucleotide;

b) detecting the presence of said label;

c) dehybridizing said first and second detection probes from said identifier probes; and

d) associating the label with the identity of a nucleotide in the candidate sequence.

34. The method of claim 33, wherein said first and second subpopulations of identifier probes are attached to beads.

35. The method of claim 34, wherein said beads are randomly distributed on the surface of the substrate.

36. The method of claim 33, wherein said candidate sequence comprises genomic DNA.

37. The method of claim 33, wherein said candidate sequence comprises a copy of a genomic DNA fragment.

38. The method of claim 33, wherein said identifier probes are prepared by an amplification technique.

39. The method of claim 33, wherein said variable region comprises two detection nucleotides.

40. The method of claim 39, wherein the two detection nucleotides are internal nucleotides of the variable region.

41. The method of claim 39, wherein the first of the two detection nucleotides is at an end of the variable region.

42. The method of claim 41, wherein the second of the two detection nucleotides is immediately adjacent to the first detection nucleotide.

43. The method of claim 42, wherein said variable region further comprises a plurality of random nucleotides immediately adjacent to the second detection nucleotide.

44. The method of claim 43, wherein said plurality of random nucleotides consists of three random nucleotides.

45. The method of claim 44, wherein said variable region further comprises a plurality of universal bases immediately adjacent to the plurality of random nucleotides.

46. The method of claim 45, wherein said plurality of universal bases consists of three universal bases.

47. The method of claim 33, wherein said variable region comprises a single detection nucleotide.

48. The method of claim 47, wherein the single detection nucleotide is an internal nucleotide of the variable region.

49. The method of claim 47, wherein the single detection nucleotide is at an end of the variable region.

50. The method of claim 49, wherein said variable region further comprises a plurality of random nucleotides immediately adjacent to the single detection nucleotide.

51. The method of claim 50, wherein said plurality of random nucleotides consists of at least two but not more than five random nucleotides.

52. The method of claim 51, wherein said variable region further comprises a plurality of universal bases immediately adjacent to the plurality of random nucleotides.

53. The method of claim 52, wherein said plurality of universal bases consists of at least two but not more than five universal bases.

54. The method of claim 33, wherein said label is located at one end of said variable region.

55. The method of claim 33, wherein said label is located at the detection position.

56. The method of claim 33, wherein the candidate sequence comprises no more than 1000 nucleotides.

57. The method of claim 56 further comprising detecting a sufficient number of labels to permit identification of at least 20 consecutive nucleotides of the candidate sequence prior to associating said labels with the identity of a nucleotide in the candidate sequence.

58. The method of claim 33, wherein said plurality of detection probes further comprises a priming sequence complementary to a portion of the primer sequence.

59. The method of claim 58, wherein said variable region comprises two detection nucleotides.

60. The method of claim 59, wherein the first of the two detection nucleotides is immediately adjacent to said priming sequence.

61. The method of claim 60, wherein the second of the two detection nucleotides is immediately adjacent to the first detection nucleotide.

62. The method of claim 58, wherein said variable region comprises a single detection nucleotide.

63. The method of claim 62, wherein the single detection nucleotide is immediately adjacent to said priming sequence.