University of California

Adam R Sciambi

Adam R Abate

Dennis Jay Eastburn

Freeman Lan

John R Haliburton

Russell S Cole

Zev Jordan Gartner

Shaun Lim

Dennis Jay Eastbum

Russell Cole

Microfluidic methods for barcoding nucleic acid target molecules to be analyzed, e.g., via nucleic acid sequencing techniques, are provided. Also provided are microfluidic, droplet-based methods of preparing nucleic acid barcodes for use in various barcoding applications. The methods described herein facilitate high-throughput sequencing of nucleic acid target molecules as well as single cell and single virus genomic, transcriptomic, and/or proteomic analysis/profiling. Systems and devices for practicing the subject methods are also provided.

Sequencing of nucleic acids via barcoding in discrete entities

Methods for the detection of components from biological samples are provided. In certain aspects, the methods may be used to detect and/or quantify specific components in a biological sample, such as tumor cells (e.g., circulating tumor cells). Systems and devices for practicing the subject methods are also provided.

Methods and systems for detecting biological components

The methods described herein, referred to as PCR-Activated Sorting (PAS), allow nucleic acids contained in biological systems to be sorted based on their sequence as detected with nucleic acid amplification techniques, e.g., PCR. The nucleic acids can be free floating or contained within living or nonliving structures, including particles, viruses, and cells. The nucleic acids can include, e.g., DNA or RNA. Systems and devices for use in practicing methods of the invention are also provided.

PCR-activated sorting (PAS)

Methods for delivering discrete entities including, e.g., cells, media or reagents to substrates are provided. In certain aspects, the methods include manipulating and/or analyzing qualities of the entities or biological components thereof. In some embodiments, the methods may be used to create arrays of microenvironments and/or for two and three-dimensional printing of tissues or structures. Systems and devices for practicing the subject methods are also provided.

High definition microdroplet printer

The disclosed embodiments generally relate to a method and system to synthesize a target molecule within a droplet. In an exemplary embodiment, a first microfluidic device configured to contact a polynucleotide-containing component from a sample with lysis reagents to form a first droplet. The lysis reagents include an enzyme having protease activity. The first droplet is encapsulated with an immiscible carrier fluid. A collection reservoir is provided to receive and incubate the first droplet for a first duration of time. The first duration of time is sufficient to inactivate the enzyme of the lysis reagent. A second microfluidic device is provided to receive the first droplet and add nucleic acid synthesis reagent to thereby form a second nucleic acid synthesis droplet in the immiscible carrier fluid. Finally, a reaction chamber is provided to synthesize the target polynucleotide within the second nucleic acid synthesis droplet.

System and method to synthesize a target molecule within a droplet

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.

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San Francisco, CA, United States of America

Adam R Sciambi