Method and system for synthesizing a target polynucleotide within a droplet

Patent metadata

Field	Value
Publication number	US-10745762-B2
Application number	US-201916279905-A
Country	US
Kind code	B2
Filing date	Feb 19, 2019
Priority date	Aug 13, 2012
Publication date	Aug 18, 2020
Grant date	Aug 18, 2020

Abstract

The disclosed embodiments relate to method, system and apparatus for synthesizing a target polynucleotide within a droplet. In an exemplary embodiment, the disclosure provides a method of synthesizing a target polynucleotide. The method includes the steps of: contacting a polynucleotide-containing component from a sample with lysis reagents in a droplet, the lysis reagents comprising an enzyme having protease activity, wherein the droplet is encapsulated with an immiscible carrier fluid; moving the droplet into a collection reservoir; incubating the droplet in the collection reservoir for a first duration and then inactivating the enzyme having protease activity; adding to the droplet a nucleic acid synthesis reagent to form a nucleic acid synthesis droplet in the immiscible carrier fluid to form a nucleic acid droplet; and synthesizing the target polynucleotide within the nucleic acid synthesis droplet.

First claim

What is claimed is:

1. A method of synthesizing a target polynucleotide, the method comprising:
contacting, on a first device, a polynucleotide-containing component from a sample with lysis reagents in a droplet, the lysis reagents comprising an enzyme having protease activity, wherein the droplet is encapsulated with an immiscible carrier fluid;
moving the droplet into a collection reservoir of a second device;
incubating the droplet in the collection reservoir for a first duration and then inactivating the enzyme having protease activity;
adding to the droplet a nucleic acid synthesis reagent to form a nucleic acid synthesis droplet in the immiscible carrier fluid to form a nucleic acid droplet; and
synthesizing the target polynucleotide within the nucleic acid synthesis droplet wherein the first and the second device are discrete; and wherein the first device and the second device are part of a microfluidic system and wherein the first device and the second device are connected by one or more fluid flow paths.

2. The method of claim 1 , wherein the first duration is a period longer than necessary for lysis.

3. The method of claim 1 , wherein the step of incubating the droplet in a collection reservoir further comprises incubating the lysate droplet and the enzyme having protease activity in the collection reservoir at a temperature sufficient to inactivate the enzyme having protease activity.

4. The method of claim 1 , wherein the step of adding to the droplet a nucleic acid synthesis reagent further comprises adding a plurality of nucleic acid synthesis reagents.

5. The method of claim 1 , wherein the nucleic acid synthesis droplet has a volume of 0.001 to 1000 picoliters.

6. The method of claim 1 , wherein the nucleic acid synthesis droplet has a diameter of between 0.1 microns to 1000 microns.

7. The method of claim 1 , wherein the step of adding a nucleic acid synthesis reagent to the droplet further comprises:
(i) contacting the lysate reagents with a continuous stream of fluid comprising the nucleic acid synthesis reagent, and
(ii) forming the nucleic acid synthesis droplet from a portion of the continuous stream of fluid and the droplet.

8. The method of claim 1 , further comprising detecting the target polynucleotide by determining a sequence of a nucleic acid synthesis product of the nucleic acid synthesis droplet or by forming a double-emulsion comprising the nucleic acid synthesis droplet within an outer droplet, and sorting the double-emulsion based on at least one of droplet size and fluorescence.

Assignees

• Univ California

Inventors

• Abate Adam R
• Eastburn Dennis Jay
• Sciambi Adam R

Classifications

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• B01F33/3011

  using a sheathing stream of a fluid surrounding a central stream of a different fluid, e.g. for reducing the cross-section of the central stream or to produce droplets from the central stream · CPC title

• B01F25/4335

  Mixers with a converging-diverging cross-section · CPC title

• B01F23/41

  Emulsifying · CPC title

• B01F33/3031

  using electro-hydrodynamic [EHD] or electro-kinetic [EKI] phenomena to mix or move the fluids · CPC title