Digital counting of individual molecules by stochastic attachment of diverse labels

• US 9,816,137 B2
• Filed: 05/19/2014
• Issued: 11/14/2017
• 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): Mahatan, Channing S
• Application Number:
  US14/281,706
• Publication Number:
  US 20150005185A1
• Time in Patent Office: 1,275 Days
• Field of Search: None
• 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 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 each primer of the plurality of primers comprises a different variable label region, and the plurality of nucleic acids comprises multiple occurrences of a target nucleic acid;

b. extending the plurality of primers attached to the plurality of nucleic acids to produce a plurality of labeled nucleic acids, wherein each one of the plurality of labeled nucleic acids comprises (i) a variable label region; and

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

c. attaching a plurality of second primers to the plurality of labeled nucleic acids and extending the plurality of second primers to produce a plurality of double-stranded labeled nucleic acids.

2. The method of claim 1, further comprising amplifying a double-stranded labeled nucleic acid of the plurality of double-stranded labeled nucleic acids to produce a first plurality of double-stranded labeled amplicons.

3. The method of claim 2, 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 a nucleic acid from the plurality of nucleic acids.

4. The method of claim 3, wherein amplifying the double-stranded labeled nucleic acid comprises conducting a polymerase chain reaction (PCR) on the double-stranded labeled nucleic acid.

5. The method of claim 3, 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.

6. The method of claim 5, 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 a nucleic acid from the plurality of nucleic acids.

7. The method of claim 6, wherein amplifying the one or more double-stranded labeled amplicons comprises conducting a polymerase chain reaction (PCR) on the one or more double-stranded labeled amplicons.

8. The method of claim 6, wherein amplifying the one or more 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.

9. The method of claim 8, wherein amplifying the one or more double-stranded labeled amplicons further comprises extending the universal primer attached to the one or more double-stranded labeled amplicons.

10. The method of claim 9, further comprising detecting a double-stranded labeled amplicon of the second plurality of double-stranded labeled amplicons.

11. The method of claim 10, 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.

12. The method of claim 10, wherein detecting the double-stranded labeled amplicon of the second plurality of double-stranded labeled amplicons comprises detecting the copy of the nucleic acid.

13. The method of claim 10, 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.

14. The method of claim 10, 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.

15. The method of claim 10, 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.

16. The method of claim 10, 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.

17. The method of claim 10, 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.

18. The method of claim 10, 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.

19. The method of claim 18, wherein the double-stranded labeled amplicon of the second plurality of labeled amplicons comprises a copy of the universal priming sequence.

20. The method of claim 19, 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.

21. The method of claim 10, 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.

22. The method of claim 10, 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.

23. The method of claim 10, wherein detecting the double-stranded labeled amplicon of the second plurality of double-stranded labeled amplicons comprises sequencing at least a portion of a junction formed between the copy of the variable label region and the copy of the nucleic acid.

24. The method of claim 1, wherein the nucleic acid of the plurality of nucleic acids is DNA.

25. The method of claim 24, wherein the DNA is genomic DNA.

26. The method of claim 1, wherein the plurality of nucleic acids is RNA.

27. The method of claim 26, wherein the RNA is mRNA.

28. The method of claim 1, wherein each primer of the plurality of primers further comprises a target specific sequence.

29. The method of claim 28, wherein the target specific sequence is a common target specific sequence.

30. The method of claim 28, wherein the target specific sequence comprises an oligodT sequence.

31. The method of claim 1, wherein each primer of the plurality of primers further comprises a sample encoder.

32. The method of claim 1, wherein attaching the plurality of primers to the plurality of nucleic acids occurs in a stochastic manner.

33. The method of claim 1, wherein attaching the plurality of primers to the plurality of nucleic acids occurs in a sequence independent manner.

34. The method of claim 1, wherein the sample is from a cell.

35. The method of claim 1, wherein the sample is from a human.

36. The method of claim 1, wherein the sample is from an organism.

37. The method of claim 1, wherein the sample is from a virus.

38. The method of claim 1, wherein each primer of the plurality of primers further comprises a universal primer sequence.

39. The method of claim 38, wherein each one of the plurality of labeled nucleic acids comprises the universal primer sequence.

40. The method of claim 38, wherein each one of the plurality of double-stranded labeled nucleic acids comprises the universal primer sequence.

41. The method of claim 40, further comprising amplifying a double-stranded labeled nucleic acid of the plurality of double-stranded labeled nucleic acids to produce a first plurality of double-stranded labeled amplicons using a universal primer that binds to the universal primer sequence.