Compositions and methods for molecular labeling

Patent metadata

Field	Value
Publication number	US-11747327-B2
Application number	US-202016928924-A
Country	US
Kind code	B2
Filing date	Jul 14, 2020
Priority date	Feb 18, 2011
Publication date	Sep 5, 2023
Grant date	Sep 5, 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 analyzing proteins of a cell, the method comprising:
segregating a single cell and barcoded binders, each barcoded binder comprising a binder linked to an oligonucleotide comprising a binder-identifying barcode, into a first droplet in an immiscible carrier fluid within a microfluidic channel, wherein the binder-identifying barcodes are sticky-ended barcodes made by a process that includes rounds of successive addition of sticky-ended partial barcodes all performed by microfluidic droplet merger;
binding the barcoded binders to proteins of the single cell;
providing a second droplet from a universal barcode droplet library into the microfluidic channel, where the second droplet includes a plurality of copies of a droplet-identifying barcode;
merging the second droplet into the first droplet;
attaching the copies of the droplet-identifying barcode to the binder-identifying barcodes of the barcoded binders bound to the proteins of the single cell to form composite barcodes;
sequencing the composite barcodes; and
identifying the binders from the sequences of the composite barcodes, thereby identifying the proteins of the single cell.

2. The method of claim 1 , wherein a number of composite barcodes sequenced quantitates the proteins of the single cell.

3. The method of claim 1 , wherein a plurality of different barcoded binders are provided to the first droplet, wherein each different barcoded binder comprises a different binder-identifying barcode and each different barcoded binder binds to a different protein of the single cell.

4. The method of claim 1 , wherein the oligonucleotide comprises a sequencing adaptor and the binder-identifying barcode.

5. The method of claim 4 , wherein the oligonucleotide further comprises a restriction site.

6. The method of claim 1 , wherein the binders of the barcoded binders are antibodies.

7. The method of claim 1 , wherein the proteins are on the surface of the cell.

8. The method of claim 1 , wherein the proteins are intracellular.

9. The method of claim 1 , wherein prior to binding the barcoded binders to the proteins of the single cell, the cell is lysed, thereby releasing at least one protein.

10. The method of claim 9 , wherein a capture-tagged binder is provided, the method further comprising binding a barcoded binder to the at least one released protein and binding the capture-tagged binder to the at least one released protein, wherein said capture-tagged binder is attached to a solid surface.

11. The method of claim 10 , wherein the capture-tagged binder is biotinylated and attached to a streptavidin-containing solid surface.

12. The method of claim 10 , wherein the solid surface is a bead.

13. The method of claim 10 , wherein the binder of the capture-tagged binder and the binder of the barcoded binder are antibodies and the released protein is an antigen.

14. The method of claim 1 , wherein each barcoded binder comprises a cleavable linker that links the binder-identifying barcode to the binder.

15. The method of claim 1 , wherein the sequencing step includes amplifying the composite barcodes using solid-phase amplification.

16. The method of claim 15 , wherein the solid-phase amplification uses forward and reverse amplification primers immobilized on a solid surface.

17. The method of claim 1 , wherein the barcoded binders bind specifically to proteins involved in cancer signaling pathways.

18. The method of claim 17 , wherein the proteins include one or more of Akt, EGF, Src, TNFRI/II, PSA, RANKL, CEA, AFP, CA125, and beta2 microglobin.

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