CA2452712A1 - Methods for detection and quantification of analytes in complex mixtures - Google Patents

Methods for detection and quantification of analytes in complex mixtures Download PDF

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Publication number
CA2452712A1
CA2452712A1 CA002452712A CA2452712A CA2452712A1 CA 2452712 A1 CA2452712 A1 CA 2452712A1 CA 002452712 A CA002452712 A CA 002452712A CA 2452712 A CA2452712 A CA 2452712A CA 2452712 A1 CA2452712 A1 CA 2452712A1
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population
nucleic acid
genedigit
target specific
genedigits
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CA002452712A
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French (fr)
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CA2452712C (en
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Krassen Dimitrov
Dwayne Dunaway
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Institute for Systems Biology
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/682—Signal amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

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 metho d of producing a population of uniquely labeled nucleic acid probes. The metho d 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 probe s 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. The method consists of (a) contacting a mixture of nucleic acid analytes under conditions sufficient for hybridization with a plurality of target specific nucleic acid probes each having a different specifier; (b) contacting the mixture under conditions sufficient for hybridization with a corresponding plurality of anti-genedigits each having a unique label, the plurality of anti-genedigits having a diversity sufficient to uniquely hybridize to genedigits within the specifiers, and (c) uniquely detecting a hybridized complex between one or more analytes in the mixture, a target specific probe, and an anti-genedigit.

Claims (87)

1. A diverse population of labels, comprising about thirty or more unique labels, wherein each of said unique labels is bound to a nucleic acid molecule.
2. The diverse population of claim 1, wherein each of said unique labels further comprises about the same unit signal, or a multiple thereof.
3. The diverse population of claim 1, wherein each of said unique labels further comprises a mixture of two or more different labels.
4. The diverse population of claim 1, further comprising unique labels having a diversity selected from a group consisting of about 40, 60, 80, 100, 120 and 140.
5. The diverse population of claim 1, further comprising about 150.
6. The diverse population of claim 1, wherein said labels are fluorescent.
7. A diverse population of uniquely labeled probes, comprising about thirty or more target specific nucleic acid probes each attached to a unique label bound to a nucleic acid.
8. The diverse population of claim 7, wherein each of said unique labels further comprises about the same unit signal, or a multiple thereof.
9. The diverse population of claim 7, wherein each of said unique labels further comprises a mixture of two or more different labels.
10. The diverse population of claim 7, further comprising a diversity of different labels selected from a group consisting of about 50, 100, 200, 500, 1,000, 2,000, 5,000, 1x10 4 and 3x10 4.
11. The diverse population of claim 7, further comprising about 1x10 5.
12. The diverse population of claim 7, wherein said labels are fluorescent.
13. The diverse population of Claim 7, wherein said target specific nucleic acid further comprises said nucleic acid bound to said unique label.
14. The diverse population of Claim 7, further comprising two attached populations of nucleic acids, one population of nucleic acids comprising said thirty or more target specific nucleic acid probes, and a second population of nucleic acids comprising said nucleic acid bound by a unique label.
15. The diverse population of Claim 7, further comprising a nucleic acid species selected from the group consisting of a specifier, an antispecifier, a genedigit, an anti-genedigit and a dendrimer.
16. A method of producing a population of labels, comprising synthesizing a population of nucleic acids, each of said nucleic acids within said synthesized population having bound a predetermined ratio of at least two different labels, wherein said population of labels has a diversity of predetermined ratios of about 30 or more.
17. The method of claim 16, wherein each of said nucleic acids bound by a predetermined ratio of at least two labels further comprises about the same unit signal.
18. The method of claim 16, wherein said diversity is selected from the group consisting of 40, 60, 80, 100, 120, 140, and 150.
19. The method of claim 16, wherein said diversity is selected from the group consisting of 200, 500, 2,000, 5,000, 1x10 4, 3x10 4 and 1x10 5.
20. The method of claim 16, wherein said labels are fluorescent.
21. The method of claim 16, wherein each of said nucleic acids within said synthesized population further comprise an anti-genedigit.
22. The method of claim 16, wherein each of said nucleic acids within said synthesized population further comprise a target specific probe.
23. A method of attaching a label to a nucleic acid probe, comprising hybridizing a nucleic acid probe having a genedigit to an anti-genedigit having a label, said genedigit comprising a set of three or more repeat sequences, said anti-genedigit comprising a cognate set of at least two complimentary repeat sequences, wherein said anti-genedigit specifically hybridizes to said genedigit through a sequence having a complexity less than the number of hybridized base pairs.
24. The method of claim 23, wherein said nucleic acid probe further comprises a specifier.
25. The method of claim 24, wherein said specifier further comprises four or more genedigits.
26. The method of claim 24, wherein said specifier further comprises five or more genedigits.
27. The method of claim 23, wherein said genedigit.
further comprises a set of four or more repeat sequences.
28. The method of claim 23, wherein said anti-genedigit further comprises a cognate set of three or more complimentary repeat sequences.
29. The method of claim 23, wherein said repeat sequences or complimentary repeat sequences further comprise about 8 nucleotides.
30. The method of claim 23, wherein said complexity is about 8.
31. The method of claim 23, wherein said number of hybridized base pairs is about 24.
32. The method of claim 23, further comprising hybridizing a population of nucleic acid probes having different genedigits to a population of different anti-genedigits each having unique labels to produce a population of uniquely labeled nucleic acid probes.
33. The method of claim 32, wherein said nucleic acid probe further comprises a specifier.
34. A method of producing a population of nucleic acid probes, comprising:
(a) producing a first population of nucleic acids comprising two or more target specific probes each having at least one genedigit, said genedigit having a set of three or more repeated sequences;
(b) producing a second population of nucleic acids comprising an anti-genedigit having a cognate set of at least two complimentary repeated sequences, and (c) hybridizing said first and second populations of nucleic acids to produce a population of target specific probes attached to an anti-genedigit, wherein said anti-genedigit hybridizes to said genedigit through a sequence having a complexity less than the number of hybridized base pairs.
35. The method of claim 34, wherein said target specific probes further comprise a specifier.
36. The method of claim 35, wherein said specifier further comprises four or more genedigits.
37. The method of claim 35, wherein said specifier further comprises five or more genedigits.
38. The method of claim 34, wherein said genedigit further comprises a set of four or more repeat sequences.
39. The method of claim 34, wherein said anti-genedigit further comprises a cognate set of three complimentary repeat sequences.
40. The method of claim 34, wherein said repeat sequences or complimentary repeat sequences further comprise about 8 nucleotides.
41. The method of claim 34, wherein said complexity is about 8.
42. The method of claim 34, wherein said number of hybridized base pairs is about 24.
43. The method of claim 34, wherein said two or more target specific probes further comprise at least one different genedigit.
44. The method of Claim 34, wherein said population of anti-genedigits further comprises two or more different anti-genedigits.
45. The method of Claim 34, wherein said anti-genedigit further comprises a label.
46. The method of claim 44, wherein said two or more different anti-genedigits further Comprise unique labels.
47. The method of claim 34, further comprising hybridizing a population of target specific probes having different genedigits to a population of different anti-genedigits each having a unique label to produce a population of uniquely labeled nucleic acid probes.
48. The method of claim 47, wherein said target specific nucleic acid probe further comprises a specifier.
49. A method of producing a population of uniquely labeled nucleic acid probes, comprising:
(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, said population having a diversity sufficient to uniquely hybridize to genedigits within said specifiers, and (c) hybridizing said populations of target nucleic acid probes to said anti-genedigits, to produce a population wherein each of said target specific probes is uniquely labeled.
50. A method of detecting a nucleic acid analyte, comprising:
(a) contacting a mixture of nucleic acid analytes with a plurality of target specific probes each attached to a unique label bound to a nucleic acid under conditions sufficient for hybridization of said probes to said target, and (b) measuring a signal from one or more said target specific probes hybridized to an analyte, wherein said signal uniquely identifies the analyte species.
51. The method of claim 50, further comprising stretching of said target specific probes hybridized to an analyte using a flow-stretch, receding meniscus, or electro-stretch technique, thereby spatially separating labels within a unique label.
52. The method of claim 50, wherein each of said unique labels further comprises about the same unit signal, or multiple thereof.
53. The method of claim 50, wherein each of said unique labels further comprises a mixture of two or more different labels.
54. The method of claim 50, wherein said plurality of target specific probes further comprises different target specific probes selected from the group consisting of about 50, 100, 200, 500, 1,000, 2,000, 5,000, 1x10 4, 3x10 4 and 1x10 5.
55. The method of claim 50, wherein said plurality of target specific probes further comprises at least one target specific probe for each nucleic acid analyte in said mixture.
56. The method of claim 50, wherein said mixture further comprises an expressed RNA population or DNA copy thereof.
57. The method of claim 50, wherein said labels are fluorescent.
58. The method of claim 50, wherein said target specific nucleic acid further comprises said nucleic acid bound to said unique label.
59. The diverse population of claim 50, further comprising two attached populations of nucleic acids, one population of nucleic acids comprising said plurality of target specific nucleic acid probes, and a second population of nucleic acids comprising said nucleic acid bound by a unique label.
60. The diverse population of claim 50, further comprising a nucleic acid species selected from the group consisting of a specifier, an antispecifier, a genedigit, an anti-genedigit and a dendrimer.
61. The method of claim 50, further comprising detecting a single copy of an analyte within said mixture.
62. A method of detecting a nucleic acid analyte, comprising:
(a) contacting a mixture of nucleic acid analytes under conditions sufficient for hybridization with a target specific probe having at least one genedigit, said genedigit having a set of three or more repeated sequences;
(b) contacting said mixture under conditions sufficient for hybridization with an anti-genedigit having a cognate set of at least two complimentary repeated sequences, and (c) detecting a hybridized complex comprising said analyte, target specific probe and said anti-genedigit, wherein said anti-genedigit hybridizes to said genedigit through a sequence having a complexity less than the number of hybridized base pairs.
63. The method of claim 62, wherein detecting said hybridized complex comprising said analyte, target specific probe and said anti-genedigit comprises the use of a flow-stretch, receding meniscus, or electro-stretch technique.
64. The method of claim 62, wherein said target specific probe further comprises a specifier.
65. The method of claim 64, wherein said specifier further comprises four or more genedigits.
66. The method of claim 64, wherein said specifier further comprises five or more genedigits.
67. The method of claim 62, wherein said genedigit further comprises a set of four or more repeat sequences.
68. The method of claim 62, wherein said anti-genedigit further comprises a cognate set of three complimentary repeat sequences.
69. The method of claim 62, wherein said repeat sequences or complimentary repeat sequences further comprise about 8 nucleotides.
70. The method of claim 62, wherein said complexity is about 8.
71. The method of claim 62, wherein said number of hybridized base pairs is about 24.
72. The method of claim 62, further comprising two or more target specific probes
73. The method of claim 72, further comprising at least one different genedigit.
74. The method of claim 72, further comprising two or more different anti-genedigits.
75. The method of claim 62, wherein said anti-genedigit further comprises a label.
76. The method of claim 74, wherein said two or more different anti-genedigits further comprise unique labels.
77. The method of claim 62, further comprising a plurality of target specific probes having different genedigits and a plurality of different anti-genedigits each having a unique label.
78. The method of claim 77, wherein said target specific probes further comprise a specifier.
79. A method of detecting a nucleic acid analyte, comprising:
(a) contacting a mixture of nucleic acid analytes under conditions sufficient for hybridization with a plurality of target specific nucleic acid probes each having a different specifier;
(b) contacting said mixture under conditions sufficient for hybridization with a corresponding plurality of anti-genedigits each having a unique label, said plurality of anti-genedigits having a diversity sufficient to uniquely hybridize to genedigits within said specifiers, and (c) uniquely detecting a hybridized complex between one or more analytes in said mixture, a target specific probe, and an anti-genedigit.
80. The method of claim 79, wherein said uniquely detecting a hybridized complex between one or more analytes in said mixture, a target specific probe, and an anti-genedigit comprises the use of a flow-stretch, receding meniscus, or electro-stretch technique.
81. A nucleic acid labeling kit, comprising a set of genedigits, a set of anti-genedigits and a unique set of labels bound to a nucleic acid.
82. The kit of claim 81, further comprising a specifier.
83. The kit of claim 81, further comprising a dendrimer.
84. The kit of claim 81, wherein said unique label further comprises a ratio of two different labels.
85. The kit of claim 81, wherein said unique label is fluorescent.
86. The kit of claim 81, further comprising a diverse population of unique labels.
87. The kit of claim 86, wherein said diversity is selected from the group consisting of 50, 100, 150, 200, 500, 1,000, 2,000, 5,000, 1x10 4, 3x10 4 and 1x10 5.
CA2452712A 2001-07-03 2002-07-03 Methods for detection and quantification of analytes in complex mixtures Expired - Lifetime CA2452712C (en)

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PCT/US2002/021278 WO2003003810A2 (en) 2001-07-03 2002-07-03 Methods for detection and quantification of analytes in complex mixtures

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