Compositions and methods for molecular labeling

Patent metadata

Field	Value
Publication number	US-12461094-B2
Application number	US-202217679310-A
Country	US
Kind code	B2
Filing date	Feb 24, 2022
Priority date	Feb 18, 2011
Publication date	Nov 4, 2025
Grant date	Nov 4, 2025

Abstract

The invention provides barcode libraries and methods of making and using them including obtaining a plurality of nucleic acid constructs in which each construct comprises a unique N-mer and a functional N-mer and segregating the constructs into a fluid compartments such that each compartment contains one or more copies of a unique construct. The invention further provides methods for digital PCR and for use of barcode libraries in digital PCR.

First claim

What is claimed is:

1 . A method for analyte detection, the method comprising the steps of:
exposing target molecules of cells from a biological sample to a plurality of antibodies comprising antibodies that are linked to a target-specific oligonucleotide, thereby forming a plurality of complexes that comprise a target molecule bound to an antibody linked to a target-specific oligonucleotide, wherein the target-specific oligonucleotide linked to an antibody in a given complex of the plurality of complexes comprises a nucleic acid that includes a target-specific sequence that identifies the target molecule bound by the antibody of that complex; isolating an individual cell comprising one or more complexes of the plurality of complexes in a compartment; attaching a compartment-specific barcode to a target-specific oligonucleotide of one or more of the complexes within the compartment; and obtaining sequence information of the compartment-specific barcode and the target-specific sequence of the one or more complexes to identify one or more of the target molecules and assign the one or more target molecules to the individual cell.

2 . The method of claim 1 , wherein the target molecules comprise a target protein.

3 . The method of claim 1 , wherein the method further comprises dissociating the target molecules from the complexes prior to obtaining the sequence information.

4 . The method of claim 1 , wherein the target molecules comprise a target antigen.

5 . The method of claim 1 , further comprising amplifying the compartment-specific barcodes prior to obtaining the sequence information.

6 . The method of claim 1 , wherein said biological sample comprises a plurality of different target molecules, and each target molecule is formed into a complex comprising the target molecule bound to an antibody linked to a target-specific oligonucleotide comprising a respective target-specific sequence.

7 . The method of claim 1 , wherein one or more of the target molecules are cell surface biomarkers.

8 . The method of claim 1 , further comprising attaching a unique molecular barcode to the target-specific oligonucleotide of one or more of the complexes within the compartment.

9 . A method for identifying a target molecule, comprising the steps of:
exposing a cell comprising a target molecule to an antibody that binds the target molecule and comprises an oligonucleotide comprising a sequence that identifies the antibody; isolating the cell in an aqueous partition with a partition barcode; attaching the partition barcode to the oligonucleotide from the antibody, forming a nucleic acid that includes the sequence that identifies the antibody and the partition barcode; obtaining sequence information of the nucleic acid that includes the sequence that identifies the antibody and the partition barcode; and based on the sequence information of the sequence that identifies the antibody, determining the identity of the target molecule, and based on the sequence information of the partition barcode, assigning the target molecule to the cell.

10 . The method of claim 9 , wherein the target molecule is a protein.

11 . The method of claim 9 , wherein the aqueous partition is a droplet formed by merging a first aqueous stream comprising the target molecule with a second aqueous stream comprising the partition barcode.

12 . The method of claim 9 , further comprising amplifying the nucleic acid prior to obtaining the sequence information.

13 . The method of claim 9 , wherein the exposing comprises exposing the cell to a plurality of different antibodies comprising antibodies that are linked to an oligonucleotide comprising a sequence that identifies the antibody to which it is linked.

Assignees

• Bio Rad Laboratories Inc

Inventors

• Samuels Michael L
• Olson Jeffrey Charles
• Watson Andrew
• Brown Keith
• Link Darren R

Classifications

• C12N15/1075 Primary

  by coupling phenotype to genotype, not provided for in other groups of this subclass · CPC title

• G01N33/532

  Production of labelled immunochemicals · CPC title

• C12Q2565/30

  Detection characterised by liberation or release of label · CPC title

• C12Q2563/179

  the label being a nucleic acid · CPC title

• C12Q2521/10

  Nucleotidyl transfering · CPC title