Granted Patent B2

US 12,551,860 · App. 17/323,902 · Granted Feb 17, 2026

Inventors: David A. Weitz (Cambridge, MA); Allon Moshe Klein (Cambridge, MA); Ilke Akartuna (Cambridge, MA); Linas Mazutis (Vilnius, LT); Marc W. Kirschner (Cambridge, MA)

Assignees: President and Fellows of Harvard College; Vilnius University

B01J19/0046B01F33/3011B01L3/502761B01L3/502776B01L3/502784C12Q1/6806B01J2219/00585B01J2219/00722B01L7/52B01L2200/0652B01L2300/021B01L2300/0663B01L2300/0858B01L2300/0867B01L2300/0883

Abstract

Claims (32)

1 . An article for barcoding nucleic acids, comprising:

a plurality of at least 10,000 monodisperse microfluidic droplets, at least 90% of the droplets encapsulating a fluid containing hydrogel particles, an oligonucleotide tag in solution, and cell lysate comprising nucleic acids,

wherein a plurality of the nucleic acids within a droplet are bound to the oligonucleotide tag,

wherein the oligonucleotide tag within the droplet is distinguishable from oligonucleotide tags within the other droplets of the plurality of 10,000 microfluidic droplets,

wherein the plurality of droplets contains an average of no more than about 1 particle/droplet,

wherein each droplet containing a hydrogel particle contains one unique oligonucleotide tag,

wherein each droplet comprises primers specific to the same unique oligonucleotide tag, which is different from oligonucleotide tags in other droplets,

wherein the oligonucleotide tag further comprises a random hexanucleotide,

wherein the random hexanucleotide is located outside a target binding region,

wherein each droplet ranges from 1 nL to 5 nL in volume,

wherein each random hexanucleotide is associated with one read;

wherein the oligonucleotide tag is a single-stranded oligonucleotide.

2 . The article of claim 1 , wherein the microfluidic droplets comprise a plurality of at least 1,000,000 distinguishable oligonucleotide tags within the microfluidic droplets.

3 . The article of claim 1 , wherein the unique oligonucleotide tag or at least some of the oligonucleotide tags each comprise a first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotides are taken from a first pool of at least 100 unique first oligonucleotides, and wherein the second oligonucleotides are taken from a second pool of at least 100 unique second oligonucleotides.

4 . The article of claim 3 , wherein the first pool comprises no more than 1,000 unique first oligonucleotides, and the second pool comprises no more than 1,000 unique second oligonucleotides.

5 . The article of claim 1 , wherein in at least about 90% of the plurality of microfluidic droplets, the cell lysate arises from only one cell.

6 . The article of claim 1 , wherein the plurality of droplets contains an average of no more than about 0.1 cells/droplet.

7 . The article of claim 1 , wherein the cell lysate comprises lysate from cancer cells.

8 . The article of claim 1 , wherein the plurality of droplets contains an average of no more than about 0.1 particles/droplet.

9 . The article of claim 1 , wherein at least some of the hydrogel particles comprise agarose.

10 . The article of claim 1 , wherein at least some of the hydrogel particles comprise polyacrylamide.

11 . The article of claim 1 , wherein at least some of the particles further comprise cleaved photocleavable linkers.

12 . The article of claim 11 , wherein the photocleavable linkers comprise acrylic phosphoramidite.

13 . The article of claim 1 , wherein the hydrogel particles have an average diameter of less than about 100 micrometers.

14 . The article of claim 1 , wherein at least some of the hydrogel particles are porous.

15 . The article of claim 1 , wherein the hydrogel particles are covalently bonded to the oligonucleotide tags via a streptavidin linkage, an amino linkage, or an acrylic phosphoramidite linkage.

16 . The article of claim 1 , wherein at least some of the oligonucleotide tags comprise a common gene sequence on an end of the oligonucleotide tag.

17 . The article of claim 1 , wherein at least some of the oligonucleotide tags comprise a poly-T sequence.

18 . The article of claim 1 , wherein the distinguishable oligonucleotide tags are separated by a Hamming distance.

19 . The article of claim 1 , wherein at least some of the oligonucleotide tags comprises a primer.

20 . The article of claim 1 , wherein the hydrogel particle contains multiple copies of the one unique oligonucleotide tag.

21 . The article of claim 1 , wherein the plurality of droplets exhibits a distribution of diameters of the droplets such that no more than about 5% of the droplets have a diameter of less than about 90% or greater than about 110% of the overall average diameter of the plurality of droplets.

Assignments (2)

Continuity (8)

References Cited (366)