Immobilization-based systems and methods for genetic analysis and other applications

Patent metadata

Field	Value
Publication number	US-12545953-B2
Application number	US-202217972442-A
Country	US
Kind code	B2
Filing date	Oct 24, 2022
Priority date	Apr 19, 2016
Publication date	Feb 10, 2026
Grant date	Feb 10, 2026

Abstract

The present invention generally relates to microfluidics and labeled nucleic acids. Certain aspects are generally directed to containing cells in gels, such as agarose gels, and determining nucleic acids within the cells, e.g., while contained within the gels. The nucleic acids may be, for example, genomic DNA, mRNA, transcriptomes, or the like. In some embodiments, for instance, both genomic DNA and RNA (e.g., as in a transcriptome) from a cell may be determined. In some cases, the nucleic acids may be attached to beads for sequencing or other purposes. Such systems may be useful, for example, for high-throughput sequencing or other applications.

First claim

What is claimed is:

1 . A method of simultaneous preparing mRNA and gDNA from a cell, comprising:
lysing cells within gel droplets to release gDNA and mRNA from the cells; fragmenting the gDNA after release from the cells; attaching the gDNA to a first set of oligonucleotides comprising a first set of barcode sequences within the gel droplets; forming cDNA from the mRNA; and attaching the cDNA to a second set of oligonucleotides comprising a second set of barcode sequences within the gel droplets, wherein the first and second sets of oligonucleotides are different.

2 . The method of claim 1 , wherein at least some of the gel droplets comprise agarose.

3 . The method of claim 1 , wherein the cells are contained within the gel droplets at no more than about 1 cell/droplet.

4 . The method of claim 1 , wherein the gel droplets have an average diameter of no more than about 1 mm.

5 . The method of claim 1 , wherein at least some of the gel droplets are contained within a microfluidic channel.

6 . The method of claim 1 , wherein fragmenting the gDNA comprises exposing the gDNA to a restriction endonuclease.

7 . The method of claim 1 , further comprising forming cDNA from the mRNA using a reverse transcriptase.

8 . The method of claim 1 , wherein attaching the gDNA to the first set of oligonucleotides comprises ligating at least some of the gDNA to the first set of oligonucleotides.

9 . The method of claim 1 , wherein attaching the cDNA to the second set of oligonucleotides comprises ligating at least some of the cDNA to the second set of oligonucleotides.

10 . The method of claim 1 , wherein the first set of barcode sequences and the second set of barcode sequences have at least one barcode sequence of at least 5 nt in common.

11 . The method of claim 1 , wherein the first set of oligonucleotides is attached to particles within the gel droplets.

12 . The method of claim 11 , wherein the second set of oligonucleotides is attached to the particles within the gel droplets.

13 . The method of claim 11 , wherein at least some of the particles comprise hydrogel particles.

14 . The method of claim 11 , wherein the particles have an average diameter of no more than about 1 mm.

15 . The method of claim 11 , wherein at least some oligonucleotides of the first set of oligonucleotides are covalently bonded to the particles.

16 . The method of claim 11 , further comprising amplifying at least some oligonucleotides of the first set of oligonucleotides and/or the second set of oligonucleotides.

17 . The method of claim 16 , further comprising encapsulating at least some of the gel droplets within a fluidic droplet prior to amplifying the oligonucleotides.

18 . The method of claim 16 , further comprising releasing at least some oligonucleotides of the first set of oligonucleotides from the particles.

19 . The method of claim 16 , further comprising bursting the fluidic droplet after amplifying the oligonucleotides.

20 . The method of claim 16 , further comprising sequencing at least some oligonucleotides of the first set of oligonucleotides and/or the second set of oligonucleotides.

Assignees

• Harvard College

Inventors

• Weitz David A
• Zhang Huidan
• Heyman John

Classifications

• B01F23/40

  Mixing liquids with liquids; Emulsifying · CPC title

• C12Q1/6827

  for detection of mutation or polymorphism · CPC title

• C12Q1/6816

  characterised by the detection means (C12Q1/6804 takes precedence) · CPC title

• C12Q1/68

  involving nucleic acids · CPC title

• B01J13/02 Primary

  Making microcapsules or microballoons {(for medical preparations A61K9/50)} · CPC title