Methods of identifying multiple epitopes in cells

• US 10,144,950 B2
• Filed: 01/31/2012
• Issued: 12/04/2018
• 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:
  US13/981,711
• Publication Number:
  US 20160251697A1
• Time in Patent Office: 2,499 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 for identifying whether a plurality of targets are present in a plurality of cells comprising:

binding to the targets in the plurality of cells a plurality of tags, wherein a tag comprises a unique binding agent (UBA) that is specific for one of the targets, and an epitope specific barcode (ESB) comprising a code that represents the target identity; and

subsequently adding multiple assayable polymer subunit (APS) oligonucleotides to each of the bound tags in the plurality of cells in an ordered manner during successive rounds of split pool synthesis wherein the APS oligonucleotides in each round anneal adjacently to the APS from a previous round via an annealing region, and covalently linking the adjacently annealed APS oligonucleotides to each other to create unique codes that represent the identities of individual cells in which the tags are bound, and wherein the method does not include a step of isolating each cell in the plurality of cells.

2. The method of claim 1, wherein the UBA comprises an antibody.

3. The method of claim 1, wherein the ESB further comprises a common linker.

4. The method of claim 1, wherein the ESB comprises a nucleic acid.

5. The method of claim 1, wherein the adjacent APSs are linked by ligation.

6. The method of claim 1, wherein the adjacent APSs are linked by Click chemistry.

7. The method of claim 1, wherein the APSs comprise an amplification primer binding region.

8. The method of claim 1, wherein annealing region is within an annealing primer.

9. The method of claim 8, wherein the annealing primer comprises a loop region.