# US 12,110,536 B2 · Methods of identifying multiple epitopes in cells

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- Number: US 12,110,536 B2
- Document ID: US12110536B2
- Status: Granted
- Kind: B2
- Application number: 18/630,941
- Filed: 2024-04-09
- Published: 2024-08-22
- Granted: 2024-10-08
- Earliest claimed: 2011-01-31
- Assignees: Roche Sequencing Solutions, Inc.
- Applicants: Roche Sequencing Solutions, Inc.
- Inventors: Garry P. Nolan
- Web page: https://patentgrep.com/patent/US12110536B2

## CPC

- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6816: characterised by the detection means (C12Q1/6804 takes precedence)
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

A method of barcoding is provided. The method comprises: (a) obtaining a population of cells or cell organelles in a first volume, wherein the cells or cell organelles comprise target molecules that are associated with a first assayable oligonucleotide subunit; (b) separating the cells or cell organelles into a plurality of second volumes, wherein at least some of second volumes receive a single cell or cell organelle from the population of cells or cell organelles; and (c) associating a plurality of second assayable oligonucleotide subunits with the first assayable oligonucleotide subunit in the second volumes, wherein at least some of the second volumes each receive a different second assayable oligonucleotide subunit, to produce at least some nucleic acid molecules that comprise a first assayable oligonucleotide subunit and a second assayable oligonucleotide subunit.

## Claims

### Claim 1

A method for barcoding comprising:
(a) obtaining a population of cells or cell organelles in a first volume, wherein the cells or cell organelles comprise target molecules that are associated with a first assayable oligonucleotide subunit;
(b) separating the cells or cell organelles into a plurality of second volumes, wherein at least some of second volumes receive a single cell or cell organelle from the population of cells or cell organelles; and
(c) associating a plurality of second assayable oligonucleotide subunits with the first assayable oligonucleotide subunit in the second volumes, wherein at least some of the second volumes each receive a different second assayable oligonucleotide subunit,
to produce at least some nucleic acid molecules that comprise a first assayable oligonucleotide subunit and a second assayable oligonucleotide subunit.

### Claim 2 (depends on 1)

The method of claim 1, wherein the cells or cell organelles are fixed and/or permeabilized.

### Claim 3 (depends on 1)

The method of claim 1, wherein the first assayable oligonucleotide subunit is covalently associated with the target molecules in step (a).

### Claim 4 (depends on 1)

The method of claim 1, wherein the first assayable oligonucleotide subunit is non-covalently associated with the target molecules in step (a).

### Claim 5 (depends on 1)

The method of claim 1, wherein the first assayable oligonucleotide subunit is directly associated with the target molecules in step (a).

### Claim 6 (depends on 5)

The method of claim 5, wherein the first assayable oligonucleotide subunit is directly associated with the target molecules by hybridization.

### Claim 7 (depends on 1)

The method of claim 1, wherein the first assayable oligonucleotide subunit is indirectly associated with the target molecules in step (a).

### Claim 8 (depends on 7)

The method of claim 7, wherein the first assayable oligonucleotide subunit is indirectly associated with the target molecules via a binding agent.

### Claim 9 (depends on 8)

The method of claim 8, wherein the binding agent is an antibody, aptamer, oligonucleotide, or primer.

### Claim 10 (depends on 1)

The method of claim 1, wherein the first assayable oligonucleotide subunit is part of a nucleic acid that is hybridized to a target molecule.

### Claim 11 (depends on 10)

The method of claim 10, wherein the nucleic acid that is hybridized to the target molecule is cDNA.

### Claim 12 (depends on 11)

The method of claim 11, wherein the cDNA is made by reverse transcription.

### Claim 13 (depends on 1)

The method of claim 1, wherein the second assayable oligonucleotide subunits are covalently associated with the first assayable oligonucleotide subunit in step (c).

### Claim 14 (depends on 1)

The method of claim 1, wherein the second assayable oligonucleotide subunits are non-covalently associated with the first assayable oligonucleotide subunit in step (c).

### Claim 15 (depends on 1)

The method of claim 1, wherein the second assayable oligonucleotide subunits are directly associated with the first assayable oligonucleotide in step (c).

### Claim 16 (depends on 1)

The method of claim 1, wherein the second assayable oligonucleotide subunits are indirectly associated with the first assayable oligonucleotide in step (c).

### Claim 17 (depends on 16)

The method of claim 16, wherein the second assayable oligonucleotide subunits are indirectly associated with the first assayable oligonucleotide subunits via a polynucleotide sequence.

### Claim 18 (depends on 16)

The method of claim 16, wherein the second assayable oligonucleotide subunits are indirectly associated with the first assayable oligonucleotide subunit via a binding agent.

### Claim 19 (depends on 1)

The method of claim 1, wherein the associating of step (c) is done by ligation, gap-fill/ligation, primer extension, hybridization, or any combination thereof.

### Claim 20 (depends on 1)

The method of claim 1, wherein the first and second assayable oligonucleotide subunits in a nucleic acid molecule form all or part of a cell origination barcode.

### Claim 21 (depends on 1)

The method of claim 1, further comprising lysing the cells between steps (b) and (c).

### Claim 22 (depends on 1)

The method of claim 1, further comprising, after step (c), pooling the second volumes into a third volume.

### Claim 23 (depends on 1)

The method of claim 1, wherein: step (c) comprises:
adding the plurality of second assayable oligonucleotide subunits to the plurality of second volumes, wherein at least some of the second volumes each receive a different second assayable oligonucleotide subunit, and
allowing the second assayable oligonucleotide subunits to associate with a nucleic acid that comprises a first assayable oligonucleotide subunit,
to produce at least some nucleic acid molecules that comprise a first assayable oligonucleotide subunit and a second assayable oligonucleotide subunit.

### Claim 24 (depends on 1)

The method of claim 1, wherein the target molecules are genomic DNA.

### Claim 25 (depends on 1)

The method of claim 1, wherein the target molecules are RNA, DNA, or cDNA.

### Claim 26 (depends on 1)

The method of claim 1, wherein the target molecules are proteins.

### Claim 27 (depends on 1)

The method of claim 1, wherein the target molecules are oligonucleotides.

### Claim 28 (depends on 8)

The method of claim 8, wherein the first assayable oligonucleotide subunit or binding agent is associated with at least one target molecule and/or a copy thereof.

### Claim 29

A composition comprising one or more cells or organelles in a volume containing both a first assayable oligonucleotide subunit and a second assayable oligonucleotide subunit.

### Claim 30 (depends on 29)

The composition of claim 29, wherein the cells or organelles are fixed and/or permeabilized.