Methods for detection and quantification of analytes in complex mixtures

• US 7,473,767 B2
• Filed: 07/03/2001
• Issued: 01/06/2009
• Est. Priority Date: 07/03/2001
• Status: Expired due to Term

• Current Assignee: Institute for System Biology
• Original Assignee: Institute for System Biology
• Inventors: Dimitrov, Krassen
• Primary Examiner(s): Chunduru; Suryaprabha
• Application Number:
  US09/898,743
• Publication Number:
  US 20030013091A1
• Time in Patent Office: 2,744 Days
• Field of Search: 536/24.31, 536/24.32, 536/22.1, 536/23.1, 436/519, 436/546, 436/501, 435/6
• US Class Current: 536/23.1
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 1/6888 for detection or identification of organisms
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/102 Multiple non-interacting labels
• CPC Class Codes: C12Q 2565/1025 labels being on the same oligonucleotide

Abstract

The invention provides a diverse population of uniquely labeled probes, containing about thirty or more target specific nucleic acid probes each attached to a unique label bound to a nucleic acid. Also provided is a method of producing a population of uniquely labeled nucleic acid probes. The method consists of (a) synthesizing a population of target specific nucleic acid probes each having a different specifier; (b) synthesizing a corresponding population of anti-genedigits each having a unique label, the population having a diversity sufficient to uniquely hybridize to genedigits within the specifiers, and (c) hybridizing the populations of target nucleic acid probes to the anti-genedigits, to produce a population in which each of the target specific probes is uniquely labeled. Also provided is a method of detecting a nucleic acid analyte.

Claims

1. A diverse population of uniquely labeled probes, comprising thirty or more target-specific polynucleotide probes that each has a detectable signal that distinguishes it from the other polynucleotide probes of the population, wherein each of said polynucleotide probes comprises a target-specific region and a region comprising a plurality of genedigits linked together in a unique combination, each genedigit being of predetermined nucleotide sequence, wherein at least two of said genedigits have different nucleotide sequences, wherein each different genedigit is attached to a corresponding anti-genedigit having a unique label monomer or a unique combination of label monomers, and wherein said population is in solution.

6. The diverse population of claim 1, wherein each said polynucleotide probes is noncovalently attached to an unlabeled bridging nucleic acid.

7. The diverse population of claim 1, wherein the polynucleotide probe is a synthetic nucleic acid molecule.

11. The diverse population of claim 6, wherein the polynucleotide probe is noncovalently attached to a target molecule.

12. The diverse population of claim 1, wherein the polynucleotide probe is noncovalently attached to a target molecule.

16. The diverse population of claim 7, wherein the poynucleotide probe is noncovalently attached to a target molecule.

20. The diverse population of any one of claims 1-5, wherein each said genedigit and each said corresponding antigenedigit are attached to one another noncovalently via hybridization.

21. The diverse population of any one of claims 1-5, wherein each of at least two of said genedigits comprises a repeated core element.

22. The diverse population of any one of claims 1-5, wherein said plurality of said genedigits is at least four genedigits, said at least four genedigits being each attached to a respective anti-genedigit.

23. The diverse population of any one of claims 1-5, wherein said plurality of said genedigits is at least five genedigits, said at least five genedigits being each attached to a respective anti-genedigit.

24. The diverse population of any one of claims 1-5, wherein at least one label monomer is light-emitting.

25. The diverse population of claim 24, wherein said label monomer is fluorescent.

26. The diverse population of any one of claims 1-5, wherein each of said uniquely labeled probes comprises a mixture of two or more different label monomers.

27. The diverse population of any one of claims 1 or 3-5, wherein the target-specific region in each unique label is different.

28. The diverse population of any one of claims 1-5, wherein at least one label monomer is a quantum dot.

29. The diverse population of any one of claims 1-5, wherein at least one anti-genedigit is a dendrimer.

30. The diverse population of claim 29, wherein the dendrimer is a fork-like dendrimer.

31. The diverse population of claim 29, wherein the dendrimer is a comb-like dendrimer.

32. The diverse population of any one of claims 1-5, wherein each said anti-genedigit is covalently attached to each said at least one label monomer.

33. The diverse population of claim 32, wherein each said at least one label monomer is fluorescent.

37. The diverse population of any one of claims 1-5, comprising 40 or more unique labels.

38. The diverse population of claim 37, comprising 100 or more unique labels.

39. The diverse population of claim 38, comprising 150 or more unique labels.

40. The diverse population of claim 39, comprising 200 or more unique labels.

41. The diverse population of claim 40, comprising 500 or more unique labels.

42. The diverse population of claim 41, comprising 1,000 or more unique labels.

43. The diverse population of claim 42, comprising 2,000 or more unique labels.

44. The diverse population of claim 43, comprising 5,000 or more unique labels.

45. The diverse population of claim 44, comprising 1×

10⁴ or more unique labels.

2. A diverse population of uniquely labeled probes, comprising thirty or more target-specific polynucleotide probes that each have a detectable signal that distinguishes it from the other polynucleotide probes of the population, wherein each of said polynucleotide probes comprises:

(i) a region comprising a plurality of genedigits linked together in a unique combination, each genedigit being of predetermined nucleotide sequence, wherein at least two of said genedigits have a different nucleotide sequence, wherein each different genedigit is attached to a corresponding anti-genedigit having a unique label monomer or a unique combination of label monomers; and

(ii) a target-specific region comprising a target-specific nucleotide sequence, said target-specific nucleotide sequence being non-covalently attached to an unlabeled bridging nucleic acid,

and wherein each unique label comprises a different target-specific region.

8. The diverse population of claim 2, wherein the polynucleotide probe is a synthetic nucleic acid molecule.

13. The diverse population of claim 2, wherein the polynucleotide probe is noncovalently attached to a target molecule.

3. A diverse population of uniquely labeled probes, comprising thirty or more target-specific polynucleotide probes that each has a detectable signal that distinguishes it from the other polynucleotide probes of the population, wherein each of said polynucleotide probes comprises a target-specific region and a region comprising a plurality of genedigits linked together in a unique combination, each genedigit being of predetermined nucleotide sequence, wherein at least two of said genedigits have a different nucleotide sequence, wherein each different genedigit is attached to a corresponding anti-genedigit having a unique label monomer or a combination of unique label monomers, wherein each said polynucleotide probe is noncovalently attached to an unlabeled bridging nucleic acid.

9. The diverse population of claim 3, wherein the polynucleotide probe is a synthetic nucleic acid molecule.

10. The diverse population of claim 3, wherein the polynucleotide probe is noncovalently attached to a target molecule.

17. The diverse population of any one of claims 10-16, wherein the target molecule is a DNA molecule and wherein said noncovalent attachment is via hybridization.

18. The diverse population of any one of claims 10-16, wherein the target molecule is unlabeled.

19. The diverse population of claim 17, wherein the target molecule is unlabeled.

34. The diverse population of any one of claims 10-16, wherein each said target molecule is attached to a chip, microarray or bead.

35. The diverse population of claim 17, wherein each said target molecule is attached to a chip, microarray or bead.

36. The diverse population of claim 19, wherein each said target molecule is attached to a chip, microarray or bead.

71. The diverse population of any one of claims 2-5, 10, and 12-16, wherein the labels are spread on a two-dimensional surface.

4. A diverse population of uniquely labeled probes, comprising thirty or more target-specific polynucleotide probes that each has a detectable signal that distinguishes it from the other polynucleotide probes of the population, wherein each of said polynucleotide probes comprises a synthetic nucleic acid molecule, said synthetic nucleic acid molecule comprising (i) a region comprising a plurality of genedigits linked together in a unique combination, each genedigit being of predetermined nucleotide sequence, wherein at least two of said genedigits have a different nucleotide sequence, wherein each different genedigit is attached to a corresponding anti-genedigit having a unique label monomer;

and (ii) a target-specific region comprising a target-specific nucleotide sequence.

14. The diverse population of claim 4, wherein the polynucleotide probe is noncovalently attached to a target molecule.

5. A diverse population of uniquely labeled probes, comprising thirty or more target-specific polynucleotide probes that each has a detectable signal that distinguishes it from the other polynucleotide probes of the population, wherein each of said polynucleotide probes comprises a target-specific region and a region comprising a plurality of genedigits linked together in a unique combination, each genedigit being a DNA of predetermined nucleotide sequence, wherein at least two of said genedigits have a different nucleotide sequence, wherein each different genedigit is attached to a corresponding anti-genedigit having a unique label monomer, each said anti-genedigit being an RNA.

15. The diverse population of claim 5, wherein the polynucleotide probe is noncovalently attached to a target molecule.

46. A diverse population of uniquely labeled probes, comprising thirty or more target-specific polynucleotide probes that each have a detectable signal that distinguishes it from the other labels of the population, wherein each of said polynucleotide probes comprises a target-specific region and a region comprising a plurality of genedigits linked together in a unique combination, each genedigit being of predetermined nucleotide sequence, wherein at least two of said genedigits have a different nucleotide sequence, wherein each different genedigit is attached to a corresponding anti-genedigit having a unique label monomer or a unique combination of label monomers, and wherein said label monomer is a quantum dot.

47. A diverse population of uniquely labeled probes, comprising 100 or more target-specific polynucleotide probes that each have a detectable signal that distinguishes it from the other labels of the population, wherein each of said polynucleotide probes comprises (i) a region comprising at least four genedigits linked together in a unique combination, each genedigit being of predetermined nucleotide sequence, wherein said at least four genedigits have a different sequence, wherein are different genedigit is noncovalently hybridized to a corresponding anti-genedigit having a unique label monomer or a unique combination of label monomers;

and (ii) a target-specific nucleotide sequence, said target-specific nucleotide sequence being noncovalently hybridized to an unlabeled target molecule, wherein each label comprises a different target-specific nucleotide sequence.

48. The diverse population of claim 47, wherein each said anti-genedigit is covalently attached to each said at least one label monomer.

49. The diverse population of claim 47, wherein said at least one label monomer is fluorescent.

50. The diverse population of any one of claims 47-49, wherein each said nucleic acid molecule is noncovalently attached via hybridization to an unlabeled bridging nucleic acid.

51. The diverse population of one of claims 47-49, wherein each said unlabeled target molecule is attached to a chip, microarray or bead.

52. A labeling kit, said kit comprising (i) in a first container, thirty or more unique polynucleotide molecules, each said polynucleotide molecule comprising a plurality of genedigits linked together in a unique combination, each genedigit being of predetermined nucleotide sequence, wherein at least two of said genedigits have a different sequence, and (ii) in one or more other containers, a plurality of corresponding anti-genedigits, each said corresponding anti-genedigit being attached to a unique label monomer or a unique combination of label monomers.

53. The labeling kit of claim 52, wherein each of at least two of said genedigits comprises a repeated core element.

54. The labeling kit of claim 52, wherein at least one label monomer is light-emitting.

55. The labeling kit of claim 54, wherein said label monomer is fluorescent.

56. The labeling kit of claim 52, wherein at least one label monomer is a quantum dot.

57. The labeling kit of claim 52, wherein at least one anti-genedigit is a dendrimer.

58. The labeling kit of claim 57, wherein the dendrimer is a fork-like dendrimer.

59. The labeling kit of claim 57, wherein the dendrimer is a comb-like dendrimer.

60. The labeling kit of claim 52, wherein each molecule further comprises a target-specific nucleotide sequence.

61. The labeling kit of claim 52, wherein each molecule is noncovalently attached to an unlabeled bridging nucleic acid.

62. The labeling kit of claim 52, comprising 40 or more unique molecules.

63. The labeling kit of claim 62, comprising 100 or more unique molecules.

64. The labeling kit of claim 63, comprising 150 or more unique molecules.

65. The labeling kit of claim 64, comprising 200 or more unique molecules.

66. The labeling kit of claim 65, comprising 500 or more unique molecules.

67. The labeling kit of claim 66, comprising 1,000 or more unique molecules.

68. The labeling kit of claim 67, comprising 2,000 or more unique molecules.

69. The labeling kit of claim 68, comprising 5,000 or more unique molecules.

70. The labeling kit of claim 69, comprising 1×

10⁴ or more unique molecules.