Digital counting of individual molecules by stochastic attachment of diverse label-tags

• US 9,315,857 B2
• Filed: 12/15/2011
• Issued: 04/19/2016
• Est. Priority Date: 12/15/2009
• Status: Active Grant

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Cellular Research, Inc. (Becton, Dickinson & Co)
• Inventors: Fu, Glenn K., Fodor, Stephen P. A.
• Primary Examiner(s): Gussow, Anne
• Assistant Examiner(s): Mahatan, Channing S
• Application Number:
  US13/327,526
• Publication Number:
  US 20130116130A1
• Time in Patent Office: 1,587 Days
• US Class Current: 1/1
• 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 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• 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 2537/16 Assays for determining copy number or wherein the copy number is of special importance
• CPC Class Codes: C12Q 2565/102 Multiple non-interacting labels

Abstract

Compositions, methods and kits are disclosed for high-sensitivity counting of individual molecules by stochastic labeling of a identical molecules in mixtures of molecules by attachment of a unique label-tags from a diverse pool of label tags to confer uniqueness to otherwise identical or indistinguishable events. Individual occurrences of target molecules randomly choose from a non-depleting reservoir of diverse label-tags. Labeled molecules may be detected by hybridization or sequencing based methods. Molecules that would otherwise be identical in information content are labeled to create a separately detectable product that can be distinctly detected. The disclosed stochastic transformation methods reduce the problem of counting molecules from one of locating and identifying identical molecules to a series of binary digital questions detecting whether preprogrammed label-tags are present. The methods may be used, for example, to count a given species of molecule within a sample.

Claims

1. A method comprising:

a) attaching a plurality of diverse label-tags to a nucleic acid target from a sample that contains multiple copies of the nucleic acid target, thereby producing a plurality of labeled targets, wherein;

i) a label-tag of the plurality of diverse label-tags comprises nucleotides selected from purine bases, pyrimidine bases, natural nucleotide bases, chemically modified nucleotide bases, biochemically modified nucleotide bases, non-natural nucleotide bases; and

ii) a labeled target of the plurality of labeled targets comprises a distinct label-tag and at least a portion of a nucleic acid target, or its complementary sequence;

b) amplifying the plurality of labeled-targets to produce a plurality of amplified labeled-targets, wherein an amplified labeled-target of the plurality of amplified labeled-targets comprises a copy of at least a portion of the nucleic acid target, or its complementary sequence, and a copy of the label-tag; and

c) detecting the plurality of amplified labeled-targets by sequencing at least a portion of the target and the label-tag; and

d) determining the number of copies of the nucleic acid target, as indicated by the number of different label-tags that are associated with the nucleic acid target.

2. The method of claim 1, wherein the label-tag comprises at least 8 nucleotides, wherein each of the at least 8 nucleotides are selected from the group consisting of purine bases, pyrimidine bases, natural nucleotide bases, chemically modified nucleotide bases, biochemically modified nucleotide bases, and non-natural nucleotide bases.

3. The method of claim 2, wherein the label-tag comprises from at least 2 to at least 20 nucleotides, wherein each of nucleotides are selected from the group consisting of purine bases, pyrimidine bases, natural nucleotide bases, chemically modified nucleotide bases, biochemically modified nucleotide bases, and non-natural nucleotide bases.

4. The method of claim 1, wherein the label-tag further comprises a universal primer sequence.

5. The method of claim 4, wherein amplifying comprises use of primers that bind to the universal primer sequence of the label-tag.

6. The method of claim 1, wherein the amplifying comprises polymerase chain reaction (PCR).

7. The method of claim 6, wherein PCR comprises balanced PCR.

8. The method of claim 6, wherein PCR comprises rolling circle amplification.

9. The method of claim 1, wherein the nucleic acid target is DNA.

10. The method of claim 1, wherein the nucleic acid target is RNA.

11. The method of claim 1, further comprising enriching the sample for the nucleic acid target.

12. The method of claim 1, wherein the sample comprises polynucleotides from tumor cells.

13. The method of claim 1, wherein the sample comprises polynucleotides from bacteria.

14. The method of claim 1, wherein the attaching step comprises ligating the plurality of label-tags to the multiple copies of the nucleic acid target.

15. The method of claim 1, wherein the attaching step comprises primer extension of the plurality of label-tags hybridized to the multiple copies of the nucleic acid target.

16. The method of claim 1, wherein the attaching step occurs on both ends of the nucleic acid target.

17. The method of claim 1, wherein the attaching step occurs in a stochastic manner.

18. The method of claim 1, wherein the attaching step occurs in a sequence independent manner.

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

20. The method of claim 19, wherein the sample comprises polynucleotides from tumor cells.

21. The method of claim 1, wherein the nucleic acid target comprises fragmented nucleic acids.