Methods for detection and quantification of analytes in complex mixtures
View Patent ↗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.
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.
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.
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.
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.
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.
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.
8. The diverse population of claim 2 , wherein the polynucleotide probe is a synthetic nucleic acid molecule.
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.
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.
13. The diverse population of claim 2 , wherein the polynucleotide probe is noncovalently attached to a target molecule.
14. The diverse population of claim 4 , wherein the polynucleotide probe is noncovalently attached to a target molecule.
15. The diverse population of claim 5 , 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.
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.
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.
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.
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 1×10 4 or more unique labels.
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 1×10 4 or more unique molecules.
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.