IP Library › Granted Patent US 12,571,038
Granted Patent B2
US 12,571,038 · App. 18/643,870 · Granted Mar 10, 2026

Digital counting of individual molecules by stochastic attachment of diverse labels

Inventors: Stephen P.A. Fodor (San Jose, CA); Glenn K. Fu (San Jose, CA)
Assignee: BECTON, DICKINSON AND COMPANY
C12Q1/6874C12N15/1065C12Q1/6809C12Q1/6837C12Q1/6851C12Q1/686C12Q1/6869C12Q1/6876G16B25/00G16B25/20G16B30/00G16B25/10
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Quick Facts
Patent No.
US 12,571,038
App. No.
18/643,870
Granted
Mar 10, 2026
Kind
B2
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 (23)

1 . A method, comprising:

(a) attaching by a sequence independent manner at least 100 first target molecules and at least 100 second target molecules in a sample with a set of diverse labels, thereby generating at least 100 new first target molecules and at least 100 new second target molecules, respectively, wherein the set comprises labels with m different label sequences, wherein said at least 100 new first molecules and said at least 100 new second molecules comprise copies of the first target molecules and copies of the second target molecules, respectively, and a label, wherein at least 100 of the new first molecules and the new second molecules have a label that is different from the labels on the other new first molecules and the new second molecules, respectively, wherein n 1 and n 2 are numbers of occurrences of the first target molecule and the second target molecule, respectively, and wherein the ratio of the greater of ni and n 2 to m is smaller than 0.2; and

(b) detecting at least 100 new first molecules and at least 100 new second molecules, wherein the numbers of the new first molecules and the new second molecules detected indicate the numbers of occurrences of the first molecule and the second molecule, respectively, in the sample.

2 . The method of claim 1 , wherein the attaching of step (a) comprises ligation of the labels to the at least 100 first target molecules and the at least 100 second target molecules.

3 . The method of claim 1 , wherein the attaching of step (a) comprises reverse transcription of the at least 100 first target molecules and the at least 100 second target molecules.

4 . The method of claim 1 , wherein the attaching of step (a) is performed using a reverse transcriptase, a DNA polymerase, or a combination thereof.

5 . The method of claim 1 , wherein the attaching of step (a) occurs on one end of the first target molecule and the second target molecule.

6 . The method of claim 1 , wherein the attaching of step (a) occurs on both ends of the first target molecule and the second target molecule.

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

8 . The method of claim 1 , wherein the detecting of step (b) comprises sequencing the at least 100 new first molecules and the at least 100 new second molecules, or products thereof.

9 . The method of claim 1 , wherein the detecting of step (b) comprises detecting the label sequence present on the at least 100 new first molecules and the at least 100 new second molecules, or products thereof.

10 . The method of claim 1 , further comprising performing one or more amplification steps to generate amplification products of the at least 100 new first molecules and the at least 100 new second molecules.

11 . The method of claim 10 , wherein the detecting of step (b) comprises detecting the label sequence present on amplification products of the at least 100 new first molecules and the at least 100 new second molecules.

12 . The method of claim 1 , wherein the first target molecule and/or the second target molecule comprise ribonucleic acid (RNA) or deoxyribonucleic acid (DNA).

13 . The method of claim 1 , wherein the first target molecule and/or the second target molecule comprise messenger ribonucleic acid (mRNA).

14 . The method of claim 1 , wherein the first target molecule and/or the second target molecule comprise cDNA, genomic DNA, or a fragment thereof.

15 . The method of claim 1 , wherein the first target molecule and the second target molecule have different sequences.

16 . The method of claim 1 , wherein the sample is a single cell.

17 . The method of claim 1 , wherein the ratio of the greater of n 1 and n 2 to m is smaller than 0.02.

18 . The method of claim 1 , wherein the label sequence is six or more nucleotides in length.

19 . A method, comprising:

(a) attaching by a random manner at least 100 first target molecules and at least 100 second target molecules in a sample with a set of diverse labels, thereby generating at least 100 new first target molecules and at least 100 new second target molecules, respectively, wherein the set comprises labels with m different label sequences, wherein said at least 100 new first molecules and said at least 100 new second molecules comprise copies of the first target molecules and copies of the second target molecules, respectively, and a label, wherein at least 100 of the new first molecules and the new second molecules have a label that is different from the labels on the other new first molecules and the new second molecules, respectively, wherein ni and n 2 are numbers of occurrences of the first target molecule and the second target molecule, respectively, and wherein the ratio of the greater of n 1 and n 2 to m is smaller than 0.2; and

(b) detecting at least 100 new first molecules and at least 100 new second molecules, wherein the numbers of the new first molecules and the new second molecules detected indicate the numbers of occurrences of the first molecule and the second molecule, respectively, in the sample.

Assignments (3)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Apr 29, 2024
From: FODOR, STEPHEN P.A.; FU, GLENN K.
To: CELLULAR RESEARCH, INC.
Reel/Frame 067253/0928 →
MERGER Recorded Apr 29, 2024
From: TUCSON ACQUISITION CORP.
To: CELLULAR RESEARCH, INC.
Reel/Frame 067254/0282 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Apr 29, 2024
From: CELLULAR RESEARCH, INC.
To: BECTON, DICKINSON AND COMPANY
Reel/Frame 067254/0293 →
Continuity (8)
Continuation 16551638 · Aug 26, 2019
Continuation 16219553 · Dec 13, 2018
Continuation 16038832 · Jul 18, 2018
Continuation 15224460 · Jul 29, 2016
Continuation 14281706 · May 19, 2014
Continuation 12969581 · Dec 15, 2010
Provisional Application 61286768 · Dec 15, 2009
Related Publication 20240279734A1 · Aug 22, 2024
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