DIGITAL COUNTING OF INDIVIDUAL MOLECULES BY STOCHASTIC ATTACHMENT OF DIVERSE LABELS

• US 20150141292A1
• Filed: 11/12/2014
• Published: 05/21/2015
• 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 9,290,809 B2
• Time in Patent Office: Days
• US Class Current: 506/16
• 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 composition comprising a plurality of beads, wherein individual beads comprise a plurality of attached oligonucleotides, and wherein the plurality of attached oligonucleotides comprise a set, m, of variable target label sequences capable of attaching to or hybridizing to a plurality, n, of target molecules in a stochastic manner, wherein m is greater than n, and wherein the plurality of attached oligonucleotides are capable of being used for stochastic labeling of one or more different types of target molecules.

2. The composition of claim 1, wherein the plurality of attached oligonucleotides further comprise a common sequence, which is the same for all oligonucleotides attached to a given bead.

3. The composition of claim 1, wherein the plurality of attached oligonucleotides further comprise a target-specific recognition sequence that is the same for all oligonucleotides attached to a given bead.

4. The composition of claim 1, wherein the plurality of attached oligonucleotides further comprise at least one primer sequence.

5. The composition of claim 1, wherein the target molecules are nucleic acid molecules.

6. The composition of claim 5, wherein the target molecules are DNA molecules.

7. The composition of claim 5, wherein the target molecules are RNA molecules.

8. The composition of claim 5, wherein the target molecules are mRNA molecules.

9. The composition of claim 1, wherein m is between 100 and 1,000.

10. The composition of claim 1, wherein m is between 1,000 and 10,000.

11. The composition of claim 1, wherein n has a value ranging from 1 to 100.

12. The composition of claim 1, wherein n has a value ranging from 100 to 1,500.

13. The composition of claim 1, wherein n has a value greater than 1,500.

14. The composition of claim 1, wherein the plurality of attached oligonucleotides is capable of being used for stochastic labeling of one or more different types of target molecules from a single cell.

15. The composition of claim 1, wherein the ratio m/n is greater than 5.

16. The composition of claim 2, wherein the common sequence is between 15 and 30 nucleotides in length.

17. The composition of claim 1, wherein the variable target label sequences are unique sequences of between 15 and 30 nucleotides in length.

18. The composition of claim 3, wherein the target-specific recognition sequence is between 10 and 30 nucleotides in length.

19. The composition of claim 14, further comprising the attached target molecules.

20. A kit comprising a plurality of beads, wherein individual beads comprise a plurality of attached oligonucleotides, and wherein the attached oligonucleotides comprise a set, m, of variable target label sequences capable of attaching to or hybridizing to a plurality, n, of target molecules in a stochastic manner, wherein m is greater than n, and wherein the plurality of attached oligonucleotides are capable of being used for stochastic labeling of one or more different types of target molecules from a single cell.

21. The kit of claim 20, wherein the plurality of attached oligonucleotides further comprise a common sequence, which is the same for all oligonucleotides attached to a given bead.

22. The kit of claim 20, wherein the plurality of attached oligonucleotides further comprise a target-specific recognition sequence that is the same for all oligonucleotides attached to a given bead.

23. The kit of claim 20, wherein the plurality of attached oligonucleotides further comprise at least one primer sequence.

24. The kit of claim 20, wherein the target molecules are nucleic acid molecules.

25. The kit of claim 20, wherein the target molecules are mRNA molecules.

26. The kit of claim 20, wherein m is between 100 and 1,000.

27. The kit of claim 20, wherein m is between 1,000 and 10,000.

28. The kit of claim 20, wherein the variable target label sequences are unique sequences of between 15 and 30 nucleotides in length.

29. The kit of claim 20, wherein the target recognition sequence for the plurality of attached oligonucleotides on each individual bead is comprised of two or more different target specific recognition sequences.

30. The kit of claim 20, further comprising PCR amplification and sequencing reagents.