INCREASING DYNAMIC RANGE FOR IDENTIFYING MULTIPLE EPITOPES IN CELLS

• US 20150329852A1
• Filed: 08/08/2013
• Published: 11/19/2015
• Est. Priority Date: 08/08/2012
• Status: Active Grant

• Current Assignee: Roche Sequencing Solutions, Inc. (Roche Holding AG)
• Original Assignee: Garry P. Nolan
• Inventors: NOLAN, Garry P.
• Granted Patent: US 10,174,310 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• 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 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid

Abstract

The invention provides methods, compositions, kits and devices for the detection of target molecules. In some embodiments, the invention allows for multiplexed target molecule detection.

Claims

1. A method for identifying whether a plurality of targets are in a plurality of cells comprising:

binding to the targets a plurality of tags, wherein a tag comprises a code that represents a) the target identity and b) the identity of the cell in which tag is binding, wherein at least a first pair of targets within the plurality vary in their relative abundance by more than 100-fold.

4. The method as in claim 1, wherein at least a third target varies in its relative abundance over both targets in the first pair of targets by more than 10-fold.

12. The method of claim 1, further comprising at least two different tags with the same target identity and cell identity.

13. The method of claim 12, wherein the at least two different tags amplify at varying rates with the same set of primers.

14. The method of claim 1, wherein individual cell or target separation or isolation is unnecessary for the binding step.

15. The method of claim 1, further comprising detecting the code, wherein individual cell or separation or isolation is unnecessary for the detecting step.

16. The method of claim 15, wherein detecting comprises sequencing.

17. The method of claim 1, wherein each target is a protein or a nucleic acid.

18. The method of claim 17, wherein each target is mRNA.

19. The method of claim 1, wherein the tag is a nucleic acid.

20. The method of claim 1, wherein the tag comprises a first UBA.

21. The method of claim 20, wherein the first UBA is specific for one of the targets.

22. The method of claim 21, wherein the first UBA comprises a mixture of labeled and unlabeled UBAs.

23. The method of claim 21, wherein the first UBA comprises an antibody.

24. The method of claim 1, wherein the tag comprises an ESB that codes for the target identity.

27. The method of claim 24, wherein the ESB comprises a mixture of labeled and unlabeled ESBs.

28. The method of claim 24, wherein the ESB comprises a nucleic acid.

29. The method of claim 1, wherein the tag comprises an APS.

30. The method of claim 29, wherein the APS comprises a detectably distinct coding unit.

31. The method of claim 29, wherein during the binding step multiple APSs are added to the tag in an ordered manner during successive rounds of split pool synthesis.

32. The method of claim 1, wherein the tag comprises at least 2 APSs.

33. The method of claim 29, wherein the APS comprises a nucleic acid.

34. The method of claim 1, wherein the tag comprises multiple APSs, an ESB, and a UBA linked by ligation.

35. The method of claim 34, wherein the multiple APSs, the ESB, or the UBA is capable of being linked Click chemistry.

36. The method of claim 34, wherein the APS or the ESB comprises an amplification primer binding region.

37. The method of claim 34, wherein the UBA, ESB, or APS is templatable.

2-3. -3. (canceled)

5-11. -11. (canceled)

25-26. -26. (canceled)

38. A composition comprising:

a) a first target molecule,

b) at least two different unique binding agents (UBA) specific for the first target molecule,

c) a first linkable UBA-dependent epitope specific barcode (ESB), and

d) a plurality of ordered assayable polymer subunit (APS), wherein the order of APSs is detectable.

39. The composition of claim 38, wherein the at least two different UBAs comprise at least two different primer binding regions that affect amplification at varying rates with the same set of primers.

40. The composition of claim 38, wherein the at least two different UBAs are linked to at least two different primer binding regions that affect amplification at varying rates with the same set of primers.

41. The composition of claim 38, wherein the at least two different UBAs comprise one or more binding regions that recruit an ESB or COB at varying efficiencies.

42. The composition of claim 38, wherein the at least two different UBAs comprise one or more capture regions that gets associated with a binding partner at varying efficiencies.

48. The composition according to claim 38, wherein the target molecule is selected from the group consisting of a peptide, a polypeptide, an oligopeptide, a protein, a phosphoprotein, an antibody, a nucleic acid, a peptide nucleic acid, a synthetic small molecule, a disaccharide, a trisaccharide, an oligosaccharide, a polysaccharide, a lipid, a steroid, and a phospholipid.

49. The composition according to claim 38, wherein the plurality of ordered APSs comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 APSs.

50. The composition according to claim 38, wherein the UBA, ESB, or APSs are templatable.

51. The composition according to claim 38, wherein an individual APS, ESB, or linking oligonucleotide molecule comprises a unique counter tag.

52. The composition of claim 51, wherein the unique counter tag is detectable.

43-47. -47. (canceled)

53. (canceled)

54. A kit for labeling a target molecule of a cell in a population of cells with a cell origination barcode, comprising

(a) n sets of m assayable polymer subunits (APSs) each comprising a distinct package of information, wherein the packages of information are capable of being linked in an ordered fashion;

and

(b) a plurality of target molecule-specific unique binding agents (UBAs) each linked with a UBA-specific epitope specific barcode (ESB), further comprising two subtypes of at least one UBA, wherein the two subtypes are linked with different ESBs.

56. The kit of claim 54, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

57. The kit of claim 54, wherein m is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

58. The kit of claim 54, wherein n is greater than 10.

59. The kit of claim 54, wherein m is greater than 20.

60. The kit of claim 54, wherein an individual APS, ESB, or linking oligonucleotide molecule comprises a unique counter tag.

61. The kit of claim 60, wherein the unique counter tag is detectable.

62. The kit of claim 54, wherein an APS or an ESB comprises an amplification primer binding region.

63. The kit of claim 54, further comprising a probe.

55. (canceled)

64. A method for detecting target molecules in a sample comprising:

(a) obtaining;

(i) a population of cells comprising the target molecules,

(ii) a plurality of target specific UBA mixtures, wherein at least one UBA mixture comprises labeled and unlabeled UBAs at a predetermined ratio,

(iii) a plurality of ESBs capable of associating with the UBAs, and

(iv) a plurality of round-specific APS sets, each set comprising a plurality of APSs that are detectably distinct from each other;

(b) contacting the mixtures of UBAs with said population of cells, wherein the labeled UBAs and the unlabeled UBAs bind to the target molecules;

(c) contacting the plurality of ESBs with said population of cells; and

(d) conducting split pool synthesis using the plurality of round-specific APSs.

65. The method of claim 64, wherein a first labeled UBA is preferentially associated with ESBs over a first unlabeled UBA of the same target by a factor of at least 10-fold.

66. The method of claim 65, wherein a second labeled UBA is preferentially associated with ESBs over a second unlabeled UBA of the same target by a factor of at least 10-fold.

67. The method of claim 64, wherein a first labeled UBA is preferentially associated with COBs over a first unlabeled UBA of the same target by a factor of at least 10-fold.

68. The method of claim 67, wherein a second labeled UBA is preferentially associated with COBs over a second unlabeled UBA by a factor of at least 10-fold

69. The method of claim 64, wherein a first labeled UBA is preferentially detected over a first unlabeled UBA of the same target by a factor of at least 10-fold.

70. The method of claim 69, wherein a second labeled UBA is preferentially detected over a second unlabeled UBA of the same target by a factor of at least 10-fold.

71. The method of claim 64, wherein a first labeled UBA is preferentially amplified over a first unlabeled UBA of the same target by a factor of at least 10-fold.

72. The method of claim 71, wherein a second labeled UBA is preferentially amplified over a second unlabeled UBA of the same target by a factor of at least 10-fold.

73. The method of claim 64, wherein a first labeled UBA is preferentially depleted over a first unlabeled UBA of the same target by a factor of at least 10-fold.

74. The method of claim 73, wherein a second labeled UBAs is preferentially depleted over a second unlabeled UBA of the same target by a factor of at least 10-fold.

112. The method of claim 64, further comprising generating an approximation of the amount of at least one target associated with a cell by detecting the amount of at least one labeled UBA bound to the target, and normalizing the detected amount by a factor that is based on the predetermined ratio of labeled and unlabeled UBAs in the at least one UBA mixture.

75-111. -111. (canceled)