Digital counting of individual molecules by stochastic attachment of diverse labels

• US 9,290,809 B2
• Filed: 11/12/2014
• Issued: 03/22/2016
• 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): Gussow, Anne
• Assistant Examiner(s): Mahatan, Channing S
• Application Number:
  US14/540,018
• Publication Number:
  US 20150141292A1
• Time in Patent Office: 496 Days
• US Class Current: 1/1
• 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 oligonucleotide labels, wherein each oligonucleotide label of the plurality of oligonucleotide labels comprises an oligo dT sequence, a sequencing primer binding site, a common sequence that is the same for all oligonucleotide labels of the plurality of oligonucleotide labels, and a unique label tag sequence, wherein the unique label tag sequence is selected from a set of at least m different label tag sequences.

2. The composition of claim 1, further comprising a set of n target molecules hybridized to said plurality of oligonucleotide labels.

3. The composition of claim 2, wherein the target molecules are RNA molecules.

4. The composition of claim 2, wherein the target molecules are mRNA molecules.

5. The composition of claim 2, wherein n has a value ranging from 1 to 100.

6. The composition of claim 2, wherein n has a value ranging from 100 to 1,500.

7. The composition of claim 2, wherein n has a value greater than 1,500.

8. The composition of claim 2, wherein the target molecules comprise one or more different types of target molecules from a single cell.

9. The composition of claim 2, wherein m is greater than n.

10. The composition of claim 9, wherein a ratio m/n is greater than 5.

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

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

13. The composition of claim 1, wherein the plurality of oligonucleotide labels are between 48 and 76 nucleotides in length.

14. A kit comprising a plurality of oligonucleotide labels, wherein each oligonucleotide label of the plurality of oligonucleotide labels comprises an oligo dT sequence, a sequencing primer binding site, a common sequence that is the same for all oligonucleotide labels of the plurality of oligonucleotide labels, and a unique label tag sequence, wherein the unique label tag sequence is selected from a set of at least m different label tag sequences.

15. The kit of claim 14, wherein m is between 100 and 1,000.

16. The kit of claim 14, wherein m is between 1,000 and 10,000.

17. The kit of claim 14, wherein the plurality of oligonucleotide labels are between 48 and 76 nucleotides in length.

18. The kit of claim 14, further comprising PCR amplification and sequencing reagents.