Method, systems and apparatus for single cell analysis

• US 10,501,739 B2
• Filed: 10/24/2018
• Issued: 12/10/2019
• Est. Priority Date: 10/18/2017
• Status: Active Grant

• Current Assignee: Mission Bio, Inc.
• Original Assignee: Mission Bio, Inc.
• Inventors: Eastburn, Dennis Jay
• Primary Examiner(s): Vivlemore, Tracy
• Assistant Examiner(s): Kaup, Sahana S
• Application Number:
  US16/169,959
• Publication Number:
  US 20190112595A1
• Time in Patent Office: 412 Days
• Field of Search: None
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12N 15/1075 by coupling phenotype to genotype, not provided for in other groups of this subclass
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array

Abstract

The disclosed embodiments relate to method, apparatus and system for high throughput single-cell DNA sequencing with droplet microfluidic. In an exemplary embodiment, a method for analyzing nucleic acids within a cell includes the steps of: (a) flowing individual cells together with a material capable of forming a polymer or microsphere that retains nucleic acids into a carrier fluid such that droplets are formed; (b) breaking the emulsion and collecting the microsphere hydrogels in an aqueous fluid; and (c) performing combinatorial labeling on the nucleic acids contained within the microspheres/hydrogels.

Claims

1. A method for analyzing nucleic acids within a cell, comprising:

(a) combining a plurality of molecular barcodes on a polymeric support with a plurality of cells in a carrier fluid to form a plurality of microsphere in an emulsion, wherein the carrier fluid defines a continuous phase fluid;

(b) disrupting the polymeric support to capture a plurality of nucleic acids contained within a respective plurality of cells and then re-polymerizing the support material;

(c) breaking the emulsion and collecting the microspheres in an aqueous fluid wherein each of the plurality of nucleic acids is retained within a respective one of the plurality of microspheres, each collected microsphere having a respective nucleic acid and an associated barcode;

(d) re-emulsifying the collected microspheres; and

(e) using the associated barcode to label the associated nucleic acid with a reaction.

2. The method of claim 1, wherein step (a) is performed under microfluidic control.

3. The method of claim 1, wherein step (a) is done with agitation or shaking.

4. The method of claim 1, wherein step (c) is performed under microfluidic control.

5. The method of claim 1, wherein step (c) is done with agitation or shaking.

6. The method of claim 1, wherein the polymeric support defines a polymeric solid support.