Multiple-emulsion nucleic acid amplification for FACS sorting

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Solution Overview

Problem

Existing droplet digital PCR techniques are limited by the inert oil carrier phase, which prevents detection, quantitation, and sorting of PCR reactions using methods like Fluorescence Activated Cell Sorting (FACS), as these methods are not compatible with oil-based systems.

Innovation Solution

The use of multiple-emulsion nucleic acid amplification methods, where nucleic acids are encapsulated in multiple-emulsion microdroplets or Giant Unilamellar Vesicles (GUVs) allowing for nucleic acid amplification without disrupting the emulsion integrity, enabling detection and sorting through techniques like FACS.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If aqueous-in-oil emulsions are used for droplet digital PCR, then high-throughput nucleic acid amplification is achieved, but detection and sorting compatibility is lost

Engineering Contradiction:

Improvehigh-throughput nucleic acid amplificationVSAvoiddetection and sorting compatibility

Core Design Contradiction:

ProductivityVSAdaptability or versatility

Solution Approach 1:

The patent introduces an aqueous-compatible carrier oil formulation that acts as an intermediary medium. This carrier oil is specifically engineered to be compatible with both the PCR reaction environment and FACS detection systems, allowing droplets to maintain structural integrity while enabling optical detection and sorting. The carrier oil formulation includes specific components that prevent droplet coalescence and maintain emulsion stability during FACS processing.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Reliability

If inert oil carrier phase is used in droplet PCR, then reaction stability is improved, but detection capability deteriorates

Engineering Contradiction:

Improvereaction stabilityVSAvoiddetection capability

Core Design Contradiction:

ReliabilityVSDifficulty of detecting and measuring

Solution Approach 1:

The patent modifies the physical and chemical parameters of the carrier oil phase. Specifically, the carrier oil formulation is adjusted to have appropriate viscosity, density, and optical properties that enable both stable PCR reactions and FACS detection. The refractive index and fluorescence characteristics are optimized to allow proper droplet detection and sorting while maintaining reaction stability.

Inventive Principle:

Principle #35Parameter changes

3Adaptability or versatility

If multiple-emulsion microdroplets are used, then detection and sorting become possible, but emulsion integrity maintenance becomes challenging

Engineering Contradiction:

Improvedetection and sorting capabilityVSAvoidemulsion integrity

Core Design Contradiction:

Adaptability or versatilityVSStability of the object's composition

Solution Approach 1:

The aqueous-compatible carrier oil serves as a protective intermediary that prevents direct interaction between the aqueous droplet contents and the external environment during FACS processing. This carrier oil formulation includes surfactants and stabilizing agents that maintain the emulsion interface stability while allowing optical signals to pass through for detection.

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent incorporates stabilizing agents and optimized emulsion formulation that provide protective cushioning against mechanical stresses encountered during FACS processing. The carrier oil formulation is designed to absorb and dissipate shear forces and pressure changes, preventing droplet rupture or coalescence before they can occur.

Inventive Principle:

Principle #11Beforehand cushioning (Prior cushioning)

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

Enables the detection, quantitation, and sorting of nucleic acids based on their sequence, facilitating the recovery of target nucleic acids or entities, thereby overcoming the limitations of traditional droplet digital PCR methods.

Implementation Method 1

Nucleic acid amplification techniques, such as PCR, can be performed in multiple-emulsion microdroplets and Giant Unilamellar Vesicles (GUV) without destroying the integrity of the multiple-emulsion microdroplets and GUVs

Methodology Applied

Scientific EffectPolymerase Chain Reaction (PCR):

Implementation Method 2

each multiple-emulsion microdroplet or GUV includes a first miscible phase fluid surrounded by an immiscible shell, wherein the multiple-emulsion microdroplet or GUV is positioned in a second miscible phase carrier fluid

Methodology Applied

Scientific EffectEmulsion: Emulsion

Implementation Method 3

the multiple-emulsion microdroplet or GUV becomes detectably labeled, e.g., fluorescently labeled as a result of a fluorogenic assay, such as Sybr staining of amplified DNA or TaqMan PCR

Methodology Applied

Scientific EffectFluorescence: Fluorescence

Data Source

PatentEP3253910B1Multiple-emulsion nucleic acid amplification

Publication Date: 2021.07.14 RGT UNIV OF CALIFORNIA

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Figure 3

AI summary

Multiple-emulsion nucleic acid amplification allows nucleic acids contained in biological systems to be detected, quantitated and/or 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 disclosure are also provided.