Methods of identifying multiple epitopes in cells

• US 10,626,442 B2
• Filed: 05/17/2017
• Issued: 04/21/2020
• Est. Priority Date: 01/31/2011
• Status: Active Grant

• Current Assignee: Roche Sequencing Solutions, Inc. (Roche Holding AG)
• Original Assignee: Roche Sequencing Solutions, Inc. (Roche Holding AG)
• Inventors: Nolan, Garry P.
• Primary Examiner(s): Dines, Karla A
• Application Number:
  US15/597,917
• Publication Number:
  US 20170321251A1
• Time in Patent Office: 1,070 Days
• Field of Search: 506 9
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/185 incorporating bases where the precise position of the bases in the nucleic acid string is important
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2525/205 Aptamer
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array

Abstract

The invention provides methods, compositions, kits and devices for the detection of target molecules. In some embodiments, the invention allows for multiplexed target molecule detection.

Claims

1. A method of uniquely labeling target molecules within a plurality of cells, the method comprising:

(a) coupling a common linker sequence to target molecules within the plurality of cells;

(b) dividing the plurality of cells into at least two primary reaction volumes, the at least two primary reaction volumes comprising a first primary reaction volume and a second primary reaction volume;

(c) providing primary nucleic acid tags to the at least two primary reaction volumes, wherein the primary nucleic acid tags provided to the first reaction volume are different from the primary nucleic acid tags provided to a second reaction volume;

(d) coupling the common linker sequences within each of the at least two primary reaction volumes with the provided primary nucleic acid tags;

(e) pooling the at least two primary reaction volumes;

(f) splitting the combined primary reaction volumes into at least two secondary reaction volumes, the at least two secondary reaction volumes comprising a first secondary reaction volume and a second secondary reaction volume;

(g) providing secondary nucleic acid tags to each of the at least two secondary reaction volumes, wherein the secondary nucleic acid tags provided to the first secondary reaction volume are different from the secondary nucleic acid tags provided to the second reaction volume; and

(h) coupling the target molecules within each of the at least two secondary reaction volumes with the provided secondary nucleic acid tags.

2. The method of claim 1, wherein the target molecules are cell-surface molecules.

3. The method of claim 1, further comprising:

(i) repeating steps (e), (f), (g), and (h) with subsequent aliquots.

4. The method of claim 3, wherein step (i) is repeated a number of times sufficient to generate a unique series of nucleic acid tags for the molecules in a single cell.

5. The method of claim 4, wherein the number of times is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or greater than 20.

6. The method of claim 1, further comprising:

fixing and permeabilizing the plurality of cells prior to step (a).

7. The method of claim 1, wherein each of the nucleic acid tags comprises:

a first nucleic acid strand comprising an annealing region at both the 3′

end and the 5′

end of a detectably distinct coding unit; and

a second template nucleic acid strand comprising;

a first annealing region that is complementary to at least one of the annealing region at the 5′

end of the first nucleic acid strand and the annealing region at the 5′

end of the adapter sequence; and

a second annealing region that is complementary to the annealing region at the 3′

end of the first nucleic acid strand.

8. The method of claim 1, further comprising generating complementary DNAs (cDNAs) within the plurality of cells.

9. The method of claim 1, wherein the target molecules are selected from at least one of RNA, cDNA, DNA, protein, peptide, and antigen.

10. The method of claim 9, wherein the target molecules are RNA, wherein the common linker sequence is a single-stranded nucleic acid molecule, and wherein step (a) comprises one of ligating a 5′

end of the single-stranded common linker to a 3′

end of the RNA or ligating a 3′

end of the single stranded common linker sequence to a 5′

end of the RNA.

11. The method of claim 9, wherein the target molecules are RNA and wherein step (a) comprises hybridizing the common linker to the RNA.

12. The method of claim 9, wherein the target molecules are DNA or DNA fragments, wherein step (a) comprises ligating the adapter sequence to the DNA.

13. The method of claim 9, wherein the target molecules are protein, peptide, or antigen, wherein the common linker sequence is coupled to an epitope specific barcode (ESB) sequence that is couples to an antibody, the epitope specific barcode configured to uniquely identify the antibody, and wherein step (a) comprises binding the antibodies comprising the common linker sequence and the epitope specific barcode sequence to the protein, peptide, or antigen.

14. The method of claim 9, wherein the target molecules are protein, peptide, or antigen, wherein the common linker sequence is coupled to an aptamer, and wherein step (a) comprises binding the aptamer to the protein, peptide, or antigen.

15. The method of claim 1, further comprising:

ligating at least two of the nucleic acid tags that are bound to the target molecules.

16. The method of claim 15, wherein the ligation is performed within the plurality of cells while performing one or more rounds of split-pool synthesis.

17. The method of claim 1, wherein at least one of the nucleic acid tags comprises a capture region.

18. The method of claim 1, further comprising:

lysing the plurality of cells to release the target molecules from within the plurality of cells.

19. The method of claim 18, wherein the labeled target molecules from a single cell all comprise the same series of assembled nucleic acid tags.

20. The method of claim 1, wherein the plurality of cells is selected from at least one of mammalian cells, bacterial cells, and viruses.

21. A method of labeling nucleic acid molecules within a first cell, the method comprising:

(a) generating complementary DNAs (cDNAs) within a plurality of cells comprising the first cell by performing reverse transcription using a unique binding agent (UBA) that is complementary to a target RNA as a reverse transcription primer;

(b) dividing the plurality of cells into a number (n) of reaction volumes;

(c) providing a plurality of nucleic acid tags to each of the n reaction volumes, each nucleic acid tag comprising;

a first nucleic acid strand comprising an annealing region at both the 3′

end and the 5′

end of a detectably distinct coding unit; and

a second template nucleic acid strand comprising an overhang sequence, the overhang sequence comprising (i) a first annealing region that is complementary to at least one of the annealing region at the 5′

end of the first nucleic acid strand and the annealing region at the 5′

end of the unique binding agent, and (ii) a second annealing region that is complementary to the annealing region at the 3′

end of the first nucleic acid strand; and

wherein each detectably distinct coding unit of the plurality of nucleic acid tags provided into a given reaction volume is the same, and wherein a different detectably distinct coding unit is provided into each of the n reaction volumes;

(d) binding at least one of the cDNAs in each of the n reaction volumes to the nucleic acid tags;

(e) pooling then reaction volumes; and

(f) repeating steps (b), (c), (d), and (e) with the pooled reaction volumes.

22. A method of barcoding nucleic acids within a cell, the method comprising:

a) generating complementary DNAs (cDNAs) within a plurality of cells by performing reverse transcription using a unique binding agent (UBA) that is complementary to a target RNA as a reverse transcription primer;

(b) dividing the plurality of cells into at least two reaction volumes;

(c) providing a plurality of nucleic acid tags to each of the at least two reaction volumes, each nucleic acid tag comprising;

a first nucleic acid strand comprising an annealing region at both the 3′

end and the 5′

end of a detectably distinct coding unit; and

a second template nucleic acid strand comprising an overhang sequence, the overhang sequence comprising (i) a first annealing region that is complementary to at least one of the annealing region at the 5′

end of the first nucleic acid strand and the annealing region at the 5′

end of the unique binding agent, and (ii) a second annealing region that is complementary to the annealing region at the 3′

end of the first nucleic acid strand; and

wherein each detectably distinct coding unit of the plurality of nucleic acid tags provided into a given reaction volume is the same, and wherein a different detectably distinct coding unit is provided into each reaction volume;

(d) binding at least one of the cDNAs in each of the at least two reaction volumes to the nucleic acid tags;

(e) pooling the at least two reaction volumes; and

(f) repeating steps (b), (c), (d), and (e) at least once with the pooled reaction volumes.