Digital Counting of Individual Molecules by Stochastic Attachment of Diverse Labels

• US 20150005185A1
• Filed: 05/19/2014
• Published: 01/01/2015
• 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.
• Granted Patent: US 9,816,137 B2
• Time in Patent Office: 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

1-30. -30. (canceled)

31. A method of analyzing a sample comprising a plurality of nucleic acids, the method comprising:

a. attaching a plurality of primers to the plurality of nucleic acids from the sample, wherein;

i. a primer of the plurality of primers is attached to a nucleic acid of the plurality of nucleic acids;

ii. two or more primers of the plurality of primers are attached to two or more different nucleic acids of the plurality of nucleic acids;

iii. the primer of the plurality of primers comprises a variable label region and a 5′

universal primer sequence;

iv. at least two primers of the plurality of primers comprise at least two different variable label regions; and

v. the plurality of nucleic acids comprise two or more different nucleic acids;

b. extending the primer of the plurality of primers attached to the nucleic acid of the plurality of nucleic acids to produce a labeled nucleic acid, wherein the labeled nucleic acid comprises (i) the primer comprising the variable label region and the 5′

universal primer sequence; and

(ii) a complementary copy of the nucleic acid that was attached to the primer;

c. attaching a 3′

primer to a 3′

end of the labeled nucleic acid and extending the 3′

primer to produce a double-stranded labeled nucleic acid, wherein the double-stranded labeled nucleic acid comprises (i) the labeled nucleic acid of step (b); and

(ii) a complementary copy of the labeled nucleic of step (b).

32. The method of claim 31, further comprising amplifying the double-stranded labeled nucleic acid to produce a first plurality of double-stranded labeled amplicons.

33. The method of claim 32, wherein a double-stranded labeled amplicon of the first plurality of labeled amplicons comprises (i) a copy of the variable label region;

and (ii) a copy of the nucleic acid from the plurality of nucleic acids.

34. The method of claim 33, wherein amplifying comprises conducting a polymerase chain reaction (PCR) on the double-stranded labeled nucleic acid.

35. The method of claim 33, further comprising amplifying one or more double-stranded labeled amplicons of the first plurality of double-stranded labeled amplicons to produce a second plurality of double-stranded labeled amplicons.

36. The method of claim 35, wherein a double-stranded labeled amplicon of the second plurality of labeled amplicons comprises (i) a copy of the variable label region;

and (ii) a copy of the nucleic acid from the plurality of nucleic acids.

37. The method of claim 36, wherein amplifying the double-stranded labeled amplicons comprises conducting a polymerase chain reaction (PCR) on the one or more double-stranded labeled amplicons.

38. The method of claim 36, wherein amplifying the double-stranded labeled amplicons comprises attaching a universal primer to the one or more double-stranded labeled amplicons, wherein the universal primer comprises a universal priming sequence.

39. The method of claim 38, wherein amplifying the double-stranded labeled amplicons further comprising extending the universal primer attached to the one or more double-stranded labeled amplicons.

40. The method of claim 39, further comprising detecting a double-stranded labeled amplicon of the second plurality of double-stranded labeled amplicons.

41. The method of claim 40, wherein detecting the double-stranded labeled amplicon of the second plurality of double-stranded labeled amplicons comprises detecting the copy of the variable label region.

42. The method of claim 40, wherein detecting the double-stranded labeled amplicon of the second plurality of double-stranded labeled amplicons comprises detecting the copy of the nucleic acid.

43. The method of claim 40, wherein detecting the double-stranded labeled amplicon of the second plurality of double-stranded labeled amplicons comprises detecting a junction formed between the copy of the variable label region and the copy of the nucleic acid.

44. The method of claim 40, wherein detecting the double-stranded labeled amplicon of the second plurality of double-stranded labeled amplicons comprises hybridizing at least a portion of the double-stranded labeled amplicon to a solid support.

45. The method of claim 40, wherein detecting the double-stranded labeled amplicon of the second plurality of double-stranded labeled amplicons comprises hybridizing at least a portion of the copy of the variable label region of double-stranded labeled amplicon to a solid support.

46. The method of claim 40, wherein detecting the double-stranded labeled amplicon of the second plurality of double-stranded labeled amplicons comprises hybridizing at least a portion of the copy of the nucleic acid of double-stranded labeled amplicon to a solid support.

47. The method of claim 40, wherein detecting the double-stranded labeled amplicon of the second plurality of double-stranded labeled amplicons comprises hybridizing at least a portion of a junction formed between the copy of the variable label region and the copy of the nucleic acid of the double-stranded labeled amplicon to a solid support.

48. The method of claim 40, wherein detecting the double-stranded labeled amplicon of the second plurality of double-stranded labeled amplicons comprises sequencing at least a portion of the double-stranded labeled amplicon.

49. The method of claim 48, wherein the double-stranded labeled amplicon of the second plurality of labeled amplicons comprises a copy of the universal priming sequence.

50. The method of claim 49, wherein sequencing at least a portion of the double-stranded labeled amplicon comprises hybridizing a sequencing primer to the copy of the universal priming sequence of the universal primer and of the plurality of primers.

51. The method of claim 40, wherein detecting the double-stranded labeled amplicon of the second plurality of double-stranded labeled amplicons comprises sequencing at least a portion of the copy of the variable label region of double-stranded labeled amplicon.

52. The method of claim 40, wherein detecting the double-stranded labeled amplicon of the second plurality of double-stranded labeled amplicons comprises sequencing at least a portion of the copy of the nucleic acid of double-stranded labeled amplicon.

53. The method of claim 40, wherein detecting the double-stranded labeled amplicon of the second plurality of double-stranded labeled amplicons comprises hybridizing at least a portion of a junction formed between the copy of the variable label region and the copy of the nucleic acid of the double-stranded labeled amplicon.

54. The method of claim 31, wherein the nucleic acid of the plurality of nucleic acids is DNA.

55. method of claim 54, wherein the DNA is genomic DNA.

56. The method of claim 31, wherein the nucleic acid of the plurality of nucleic acids is RNA.

57. The method of claim 56, wherein the RNA is mRNA.

58. The method of claim 31, wherein the primer of the plurality of primers further comprises a target specific sequence.

59. The method of claim 58, wherein the target specific sequence is a common target specific sequence.

60. The method of claim 58, wherein the target specific sequence comprises an oligodT sequence.

61. The method of claim 31, wherein the primer of the plurality of primers further comprises a sample encoder.

62. method of claim 31, wherein attaching the plurality of primers to the plurality of nucleic acids occurs in a stochastic manner.

63. The method of claim 31, wherein attaching the plurality of primers to the plurality of nucleic acids occurs in a sequence independent manner.

64. The method of claim 31, wherein the sample is from a cell.

65. The method of claim 31, wherein the sample is from a human.

66. The method of claim 31, wherein the sample is from an organism.