Digital counting of individual molecules by stochastic attachment of diverse labels

• US 8,835,358 B2
• Filed: 12/15/2010
• Issued: 09/16/2014
• Est. Priority Date: 12/15/2009
• Status: Active Grant

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Cellular Research, Inc. (Becton, Dickinson & Co)
• Inventors: Fodor, Stephen P. A., Fu, Glenn K.
• Primary Examiner(s): Gussow, Anne
• Assistant Examiner(s): Mahatan, Channing S
• Application Number:
  US12/969,581
• Publication Number:
  US 20110160078A1
• Time in Patent Office: 1,371 Days
• US Class Current: 506/9
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• 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 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2537/157 A reaction step characterised by the number of molecules incorporated or released
• CPC Class Codes: C12Q 2565/102 Multiple non-interacting labels
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
• CPC Class Codes: G16B 25/10 Gene or protein expression profiling; Expression-ratio estimation or normalisation
• CPC Class Codes: G16B 25/20 Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids

Abstract

Compositions, methods and kits are disclosed for high-sensitivity single molecule digital counting by the stochastic labeling of a collection of identical molecules by attachment of a diverse set of labels. Each copy of a molecule randomly chooses from a non-depleting reservoir of diverse labels. Detection may be by a variety of methods including hybridization based or sequencing. Molecules that would otherwise be identical in information content can be labeled to create a separately detectable product that is unique or approximately unique in a collection. This stochastic transformation relaxes the problem of counting molecules from one of locating and identifying identical molecules to a series of binary digital questions detecting whether preprogrammed labels are present. The methods may be used, for example, to estimate the number of separate molecules of a given type or types within a sample.

Claims

1. A method for counting n wherein n is a number of molecules of a first target molecule that are present in a sample comprising:

(a) attaching by a species-independent manner to each occurrence of the first target molecule a label from a set of diverse labels, wherein the set comprises m different labels, thereby generating for each occurrence of the first target molecule, a new molecule that comprises a copy of the first target molecule and a label, wherein more than 90% of the new molecules have a label that is different from the labels on the other new molecules;

(b) detecting each new molecule by detecting each label present on the new molecule; and

(c) counting a number of new molecules, thereby counting the number of molecules of the first target molecule in the sample.

2. The method of claim 1 wherein a ratio of m to n is greater than 50.

3. The method of claim 1 wherein a ratio of m to n is greater than 100.

4. The method of claim 1 wherein a ratio of m to n is greater than 500.

5. The method of claim 1 wherein a ratio of m to n is greater than 1000.

16. The method of claim 1, wherein the attaching of step (a) is stochastic.

17. The method of claim 1, wherein the attaching of step (a) comprises ligation of the label to an occurrence of the first target molecule.

18. The method of claim 1, wherein the attaching of step (a) comprises reverse transcription of an occurrence of the first target molecule.

19. The method of claim 1, wherein the attaching of step (a) comprises use of the label for primer extension.

20. The method of claim 1, wherein the first target molecule is a nucleic acid.

21. The method of claim 1, wherein the first target molecule is RNA.

22. The method of claim 21, wherein the RNA is mRNA.

23. The method of claim 22, wherein the labels comprise an oligodT sequence.

24. The method of claim 23, wherein the labels are attached to the molecules by hybridization of the oligodT sequence of the label to a polyA tail of the mRNA.

25. The method of claim 1, wherein the first target molecule is DNA.

26. The method of claim 1, further comprising fragmenting the occurrences of the first target molecule.

27. The method of claim 26, wherein the occurrences of the first target molecule are fragmented prior to the attaching of step (a).

28. The method of claim 26, wherein fragmenting the occurrences of the first target molecule comprises restriction enzyme digestion of the occurrences of the first target molecule.

29. The method of claim 1, further comprising amplifying the new molecules.

30. The method of claim 29, wherein amplifying comprises PCR.

31. The method of claim 29, wherein amplifying comprises rolling circle amplification.

32. The method of claim 1, wherein detecting the new molecule comprises detecting at least a portion of the copy of the first target molecule.

33. The method of claim 1, wherein detecting the new molecule comprises detecting a junction formed between the label and the copy of the first target molecule.

34. The method of claim 1, wherein detecting the new molecule comprises detecting at least a portion of the copy of the first target molecule and at least a portion of the label.

35. The method of claim 1, wherein detecting the new molecule comprises hybridization of the new molecule to a solid support.

36. The method of claim 35, wherein the solid support is an array.

37. The method of claim 35, wherein the solid support comprises a plurality of probes.

38. The method of claim 37, wherein a probe of the plurality of probes comprises a sequence that is complementary to at least a portion of the copy of the first target molecule.

39. The method of claim 37, wherein a probe of the plurality of probes comprises a sequence that is complementary to at least a portion of the label.

40. The method of claim 37, wherein a probe of the plurality of probes comprises a sequence that is complementary to a junction formed between the label and the copy of the first target molecule.

41. The method of claim 37, wherein a probe of the plurality of probes comprises a sequence that is complementary to at least a portion of the label and at least a portion of the copy of the first target molecule.

42. The method of claim 1, wherein the attaching of step (a) further comprises attaching labels to occurrences of one or more additional target molecules, wherein the first target molecule is different from the one or more additional target molecules.

43. The method of claim 42, wherein the counting of step (c) further comprises counting a number of new molecules of the one or more additional target molecules, thereby counting a number of molecules of the one or more additional target molecules.

44. The method of claim 42, wherein the one or more additional target molecules comprise 2 or more target molecules.

45. The method of claim 42, wherein the one or more additional target molecules comprise 5 or more target molecules.

46. The method of claim 42, wherein the one or more additional target molecules comprise 10 or more target molecules.

47. The method of claim 42, wherein m different labels in the set of labels is constant.

48. The method of claim 42, wherein the set of labels comprises multiple copies of the same label.

49. The method of claim 48, wherein copies of a first label may attach to the occurrences of the first target molecule and the occurrences of the one or more additional target molecules.

50. The method of claim 1, wherein the attaching of step (a) occurs on one end of an occurrence of the first target molecule.

51. The method of claim 1, wherein the attaching of step (a) occurs on both ends of an occurrence of the first target molecule.

52. The method of claim 1, wherein the label comprises an adaptor.

53. The method of claim 1, wherein the label comprises a universal primer.

54. The method of claim 42, wherein the label does not comprise a sequence that is specific to one species of target molecules.

55. The method of claim 1, wherein the copy of the first target molecule is an occurrence of the first target molecule.

56. The method of claim 1, wherein the copy of the first target molecule is a copy of an occurrence of the first target molecule.

57. The method of claim 1, wherein the copy of the first target molecule is a complement or reverse complement of an occurrence of the first target molecule.

6. A method comprising:

(a) combining a mixture comprising at least two distinct target nucleic acid molecules with a pool of nucleic acid label-tags, wherein the pool of nucleic acid label-tags comprises a plurality of nucleic acid label-tags with different sequences;

(b) attaching at least two nucleic acid label-tags from the pool of nucleic acid label-tags to the at least two distinct target nucleic acid molecules to obtain at least two label-tag-target nucleic acid molecules, wherein the distinct target nucleic acid molecules have different sequences from one another;

(c) amplifying at least a portion of the label-tag-target nucleic acid molecules, wherein an amplified portion of the label-tag-target nucleic acid molecules comprises at least a portion of said target nucleic acid molecule; and

(d) detecting an amplified product of step (c).

7. The method of claim 6, wherein the attaching is stochastic.

8. The method of claim 6, wherein distinct target nucleic acid molecules are known targets.

9. The method of claim 6, wherein the nucleic acid label-tags are designed to bind to the distinct target nucleic acid molecules.

10. The method of claim 6, wherein an identity of the distinct target nucleic acid molecules is determined after the detecting step.

11. The method of claim 6, wherein the nucleic acid label-tags may bind to any of the distinct target nucleic acid molecules.

12. The method of claim 6, wherein the attaching occurs on both ends of at least one of the two distinct target nucleic acid molecules.

13. The method of claim 6, wherein the detecting comprises hybridizing at least a portion of the label-tag-target nucleic acid molecules to a solid or semi-solid substrate.

14. The method of claim 6, wherein the detecting comprises hybridizing at least a portion of the target nucleic acid molecule to a solid or semi-solid substrate.

15. The method of claim 6, wherein the detecting comprises hybridizing at least a portion of the nucleic acid label-tag to a solid or semi-solid substrate.

58. The method of claim 6, wherein the attaching of step (b) occurs in a species-independent manner.

59. The method of claim 6, wherein the attaching of step (b) occurs in a non-specific manner.

60. The method of claim 6, wherein the pool of nucleic acid label-tags comprises multiple copies of a nucleic acid label-tag.

61. The method of claim 60, wherein the copies of the nucleic acid label-tag may attach to copies of a first target nucleic molecule and copies of a second target nucleic acid molecule.

62. The method of claim 6, wherein the attaching of step (b) comprises ligation of the label-tags to copies of the two distinct target nucleic acid molecules.

63. The method of claim 62, wherein the copies of the two distinct target nucleic acid molecules are occurrences of the two distinct target nucleic acids molecules.

64. The method of claim 62, wherein the copies of the two distinct target nucleic acid molecules are copies of occurrences of the two distinct target nucleic acids molecules.

65. The method of claim 62, wherein the copies of the two distinct target nucleic acid molecules are complements or reverse complements of occurrences of the two distinct target nucleic acids molecules.

66. The method of claim 6, wherein the attaching of step (b) comprises reverse transcription of the two distinct target nucleic acid molecules.

67. The method of claim 6, wherein the two distinct target nucleic acid molecules are RNA.

68. The method of claim 67, wherein the RNA is mRNA.

69. The method of claim 68, wherein the label-tags comprise an oligodT sequence.

70. The method of claim 69, wherein the label-tags are attached to the two distinct target nucleic acid molecules by hybridization of the oligodT sequence of the label-tag to a polyA tail of the mRNA.

71. The method of claim 6, wherein the two distinct target nucleic acid molecules are DNA.

72. The method of claim 6, further comprising fragmenting the two distinct target nucleic acid molecules.

73. The method of claim 72, wherein the two distinct target nucleic acid molecules are fragmented prior to the attaching of step (b).

74. The method of claim 72, wherein fragmenting the two distinct target nucleic acid molecules comprises restriction enzyme digestion of the two distinct target nucleic acid molecules.

75. The method of claim 6, wherein amplifying comprises PCR.

76. The method of claim 6, wherein amplifying comprises rolling circle amplification.

77. The method of claim 6, wherein detecting the amplified product comprises detecting at least a portion of the distinct target nucleic acid molecule.

78. The method of claim 6, wherein detecting the amplified product comprises detecting at least a portion of the label-tag.

79. The method of claim 6, wherein detecting the amplified product comprises detecting a junction formed between the label-tag and the distinct target nucleic acid molecule.

80. The method of claim 6, wherein detecting the amplified product comprises detecting at least a portion of the distinct target nucleic acid molecule and at least a portion of the label.

81. The method of claim 6, wherein detecting the amplified product comprises hybridization of the amplified product to a solid support.

82. The method of claim 81, wherein the solid support is an array.

83. The method of claim 81, wherein the solid support comprises a plurality of probes.

84. The method of claim 83, wherein a probe of the plurality of probes comprises a sequence that is complementary to at least a portion of the distinct target nucleic acid molecule.

85. The method of claim 83, wherein a probe of the plurality of probes comprises a sequence that is complementary to at least a portion of the label-tag.

86. The method of claim 83, wherein a probe of the plurality of probes comprises a sequence that is complementary to a junction formed between the label-tag and the distinct target nucleic acid molecule.

87. The method of claim 83, wherein a probe of the plurality of probes comprises a sequence that is complementary to at least a portion of the label-tag and at least a portion of the distinct target nucleic acid molecule.

88. The method of claim 6, further comprising determining a count of the at least two distinct target nucleic acid molecules based on the detection of the amplified product.

89. The method of claim 88, wherein determining the count of the at least two distinct target nucleic acid molecules comprises determining the count of 5 or more distinct target nucleic acid molecules.

90. The method of claim 88, wherein determining the count of at least two distinct target nucleic acid molecules comprises determining the count of 10 or more distinct target molecules.

91. The method of claim 6, wherein the attaching of step (b) occurs on one end of the distinct target nucleic acid molecules.

92. The method of claim 6, wherein the attaching of step (b) occurs on both ends of the distinct target nucleic acid molecules.

93. The method of claim 6, wherein the label-tag comprises an adaptor.

94. The method of claim 6, wherein the label-tag comprises a universal primer.

95. The method of claim 6, wherein the label-tag does not comprise a sequence that is specific to one species of at least two distinct target nucleic acid molecules.