Digital counting of individual molecules by stochastic attachment of diverse labels

• US 9,708,659 B2
• Filed: 11/12/2014
• Issued: 07/18/2017
• 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:
  US14/540,029
• Publication Number:
  US 20150099673A1
• Time in Patent Office: 979 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 system for counting n, wherein n is a number of nucleic acid target molecules that are present in a single cell from a sample comprising cells, the system comprising:

a) a diverse set of labels, wherein the set comprises m different labels, wherein the ratio of m to n is greater than 5;

b) a plurality of reaction vessels for attaching a label from the diverse set of labels to each occurrence of the nucleic acid target molecules from each single cell from said sample comprising cells to generate a set of labeled nucleic acid target molecules, wherein the individual labeled nucleic acid target molecules comprise all or a portion of the complementary sequence of a nucleic acid target molecule and a label from the diverse set of labels; and

c) processing software for counting n from a number of labeled nucleic acid target molecules detected.

2. The system of claim 1, wherein said processing software uses Poisson statistics.

3. The system of claim 1, wherein the nucleic acid target molecules present in the sample are DNA molecules, and wherein the labeled nucleic acid target molecules comprise complementary sequences of the DNA molecule sequences attached to a label from the diverse set of labels.

4. The system of claim 1, wherein the nucleic acid target molecules present in the sample are mRNA molecules, and wherein the labeled nucleic acid target molecules comprise cDNA sequences of the mRNA molecule sequences attached to a label from the diverse set of labels.

5. The system of claim 1, wherein the ratio of m to n is greater than 100.

6. The system of claim 1, wherein the diverse set of labels comprises a plurality of oligonucleotides.

7. The system of claim 6, wherein the plurality of oligonucleotides are attached to one or more beads.

8. The system of claim 7, wherein each oligonucleotide of the plurality of oligonucleotides comprises a common sequence, a variable target label sequence, and a target-specific recognition sequence capable of attaching to or hybridizing to the target molecule.

9. The system of claim 8, wherein the oligonucleotides further comprise one or more primer sequences.

10. The system of claim 9, wherein a reaction vessel of the plurality of reaction vessels contains a single bead.

11. The system of claim 10, wherein the reaction vessel of the plurality of reaction vessels that contains a single bead also contains a single cell from said sample comprising cells.

12. The system of claim 11, wherein the reaction vessel of the plurality of reaction vessels is a microwell.

13. A system for counting n, wherein n is a number of nucleic acid target molecules that are present in a single cell from a sample comprising cells, the system comprising:

a) a diverse set of labels comprising a plurality of oligonucleotides attached to each bead of a plurality of beads, wherein the set of labels comprises m different labels, wherein m>

n, and wherein each oligonucleotide of the plurality of oligonucleotides comprises a common sequence, a variable target label sequence, and a target-specific recognition sequence capable of attaching to or hybridizing to the target molecule;

b) a plurality of reaction vessels comprising a single cell from said sample comprising cells and a single bead from said plurality of beads, wherein each reaction vessel is used for attaching a label from the diverse set of labels attached to the bead contained therein to each occurrence of the nucleic acid target molecules from the single cell contained therein to generate a set of labeled nucleic acid target molecules, wherein the individual labeled nucleic acid target molecules comprise all or a portion of the complementary sequence of a nucleic acid target molecule and a label from the diverse set of labels; and

c) processing software for counting n from a number of labeled nucleic acid target molecules detected.

14. The system of claim 13, wherein said processing software uses Poisson statistics.

15. The system of claim 13, wherein the nucleic acid target molecules present in the sample comprising cells are DNA molecules, and wherein the labeled nucleic acid target molecules comprise complementary sequences of the DNA molecule sequences attached to a label from the diverse set of labels.

16. The system of claim 13, wherein the nucleic acid target molecules present in the sample comprising cells are mRNA molecules, and wherein the labeled nucleic acid target molecules comprise cDNA sequences of the mRNA molecule sequences attached to a label from the diverse set of labels.

17. The system of claim 13, wherein the ratio of m to n is greater than 100.

18. The system of claim 13, wherein the oligonucleotides further comprise one or more primer sequences.

19. The system of claim 13, wherein the common sequence for the plurality of oligonucleotides attached to a given bead is different from that for the pluralities of oligonucleotides attached to other beads in the plurality of beads.

20. The system of claim 13, wherein the reaction vessel of the plurality of reaction vessels is a microwell.