Granted Patent B2

US 12,606,859 · App. 17/147,283 · Granted Apr 21, 2026

Inventors: Dennis Prosen (Foster City, CA); Katherine Lazaruk (Redwood City, CA)

Assignee: BECTON, DICKINSON AND COMPANY

C12Q1/6806C12P19/34C12Q1/6816C12Q1/6874C12Q2565/1015

Abstract

Claims (28)

1 . A method for simultaneous measurement of protein and gene expressions in cells, comprising:

contacting a plurality of cellular component-binding reagents with a plurality of cells comprising a plurality of cellular component targets and copies of a nucleic acid target,

wherein the nucleic acid target comprises mRNA,

wherein each cellular component-binding reagent of the plurality of cellular component-binding reagents comprises a cellular component-binding reagent specific oligonucleotide comprising a unique identifier for the cellular component-binding reagent and a poly(A) sequence 18-30 nucleotides in length,

wherein the cellular component-binding reagent specific oligonucleotide comprises DNA, and

wherein the cellular component-binding reagent is capable of specifically binding to at least one of the plurality of cellular component targets;

partitioning the plurality of cells associated with the cellular component-binding reagents to a plurality of partitions,

wherein a partition of the plurality of partitions comprises a single cell from the plurality of cells associated with the cellular component-binding reagents;

in the partition comprising the single cell, contacting a solid support comprising a plurality of oligonucleotide barcodes with the cellular component-binding reagent specific oligonucleotides and the copies of the nucleic acid target for hybridization,

wherein the oligonucleotide barcodes each comprise a poly(T) sequence, a first universal sequence, a first molecular label, and a cleavage region, wherein the cleavage region is 5′ of the first universal sequence and comprises one or more cleavage sites;

extending the cellular component-binding reagent specific oligonucleotides hybridized to the plurality of oligonucleotide barcodes to generate a plurality of extended cellular component-binding reagent specific oligonucleotides each comprising a complement of the first molecular label, a complement of the cleavage region, and a complement of the first universal sequence;

extending the plurality of oligonucleotide barcodes hybridized to the copies of a nucleic acid target to generate a plurality of barcoded nucleic acid molecules each comprising a poly(T) sequence, a first universal sequence, a cleavage region, a sequence complementary to the at least a portion of the nucleic acid target and the first molecular label,

wherein the extended cellular component-binding reagent specific oligonucleotides each comprise a double-stranded cleavage region and wherein the plurality of barcoded nucleic acid molecules each comprise a single-stranded cleavage region;

contacting the plurality of barcoded nucleic acid molecules and the plurality of extended cellular component-binding reagent specific oligonucleotides with one or more cleaving agents to generate:

(i) a plurality of cleaved barcoded nucleic acid molecules not associated with the solid support, and

(ii) a plurality of cleaved extended cellular component-binding reagent specific oligonucleotides associated with the solid support

separating the plurality of cleaved barcoded nucleic acid molecules from the solid support;

denaturing the cleaved extended cellular component-binding reagent specific oligonucleotides associated with the solid support to generate a plurality of denatured cleaved extended cellular component-binding reagent specific oligonucleotides;

separating the plurality of denatured cleaved extended cellular component-binding reagent specific oligonucleotides from the solid support

obtaining sequence information of the plurality of separated extended cellular component-binding reagent specific oligonucleotides, or products thereof, to determine the number of copies of at least one cellular component target of the plurality of cellular component targets in one or more of the plurality of cells; and

obtaining sequence information of the plurality of separated barcoded nucleic acid molecules, or products thereof, to determine the copy number of the nucleic acid target in one or more of the plurality of cells.

2 . The method of claim 1 , wherein the one or more cleavage sites comprise a non-canonical nucleotide, a cleaver recognition sequence, or any combination thereof.

3 . The method of claim 1 , wherein the one or more cleaving agents comprise a DNA glycosylase, an AP cleaving agent, or any combination thereof, wherein the DNA glycosylase is capable of cleaving a base portion of the non-canonical nucleotide to generate an apurinic/apyrimidinic (AP) site, and wherein the AP cleaving agent is capable of cleaving a phosphodiester backbone adjacent to the AP site.

4 . The method of claim 3 , wherein the AP cleaving agent comprises an AP cleaving enzyme, an amine, heat, basic conditions, acidic conditions, or any combination thereof, and optionally the amine comprises N,N′-dimethylethylenediamine (DMED).

5 . The method of claim 3 , wherein the AP cleaving enzyme comprises an AP lyase, an AP endonuclease, or any combination thereof.

6 . The method of claim 1 , wherein the cellular component-binding reagent specific oligonucleotide comprises a second universal sequence, and wherein obtaining sequence information of the plurality of separated extended cellular component-binding reagent specific oligonucleotides, or products thereof, comprises:

amplifying the plurality of separated extended cellular component-binding reagent specific oligonucleotides, or products thereof, using a primer capable of hybridizing to the first universal sequence, or a complement thereof, and a primer capable of hybridizing to the second universal sequence, or a complement thereof, to generate a plurality of amplified separated extended cellular component-binding reagent specific oligonucleotides; and

obtaining sequencing data of the plurality of amplified separated extended cellular component-binding reagent specific oligonucleotides, or products thereof.

Assignments (1)

Continuity (2)

References Cited (400)