Compositions and methods for molecular labeling

Patent metadata

Field	Value
Publication number	US-11768198-B2
Application number	US-202016795467-A
Country	US
Kind code	B2
Filing date	Feb 19, 2020
Priority date	Feb 18, 2011
Publication date	Sep 26, 2023
Grant date	Sep 26, 2023

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 protein detection, the method comprising:
introducing a protein-specific binding reagent linked to an oligonucleotide barcode to a sample comprising a population of cells;
encapsulating the cells in sample droplets such that each sample droplet comprises a single cell and said protein-specific binding reagent by merging droplets containing said cells and said protein-specific binding reagents; and
determining the presence of proteins of said cells that are bound to said protein-specific binding reagents.

2. The method of claim 1 , further comprising the step of lysing said cells.

3. The method of claim 1 , wherein said protein-specific binding reagent is selected from an antibody, an aptamer, a nucleic acid-labeled protein.

4. The method of claim 1 , wherein the protein-specific binding reagents are coupled to beads.

5. The method of claim 1 , wherein the protein-specific binding reagents are cleavable from the proteins to which they bind.

6. The method of claim 1 , wherein the proteins are cell-surface proteins.

7. The method of claim 1 , wherein the encapsulating step comprises loading the cells into a microfluidic chip.

8. The method of claim 1 , wherein the protein-specific binding reagents further comprise a universal binding site.

9. The method of claim 1 , further comprising quantifying the proteins.

10. The method of claim 1 , wherein the oligonucleotide barcodes linked to the protein-specific binding reagents have ligation-competent ends and the method includes ligating copies of droplet-identifying barcodes to the oligonucleotide barcodes linked to said protein-specific binding reagents.

11. The method of claim 2 , wherein lysing occurs within said droplets.

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

• C12Q2521/10

  Nucleotidyl transfering · CPC title

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• G01N33/53 Primary

  Immunoassay; Biospecific binding assay; Materials therefor · CPC title