Liquid Phase Ligation Assay for Nucleic Acid Detection

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Solution Overview

Problem

Current nucleic acid detection methods face challenges in efficiently detecting target sequences, especially in complex samples, due to issues with specificity and sensitivity, particularly when dealing with low-abundance targets and high-abundance sequences, and require multiple steps and transfers.

Innovation Solution

The method involves using a ligation assay with downstream and upstream detector oligonucleotides that hybridize to target sequences, followed by ligation and optional extension, where nucleases are strategically employed to degrade excess or non-specifically bound detectors, allowing for sensitive detection in a single reaction container without the need for solid-phase immobilization, enabling detection of nucleic acids like mRNAs and miRNAs at the single-cell level.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If multiple steps and transfers are used in nucleic acid detection, then detection completeness can be improved, but assay complexity and time consumption increase

Engineering Contradiction:

Improvedetection completenessVSAvoidassay complexity

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The patent combines multiple detection steps (hybridization, ligation, extension, and detection) into a single reaction container, eliminating the need for multiple transfers and separations. This merging of steps maintains detection completeness while significantly reducing assay complexity and time consumption.

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The reaction container serves multiple functions: it acts as both the hybridization chamber, ligation chamber, extension chamber, and detection chamber. This multi-functionality allows all detection steps to occur in one place, resolving the contradiction between detection completeness and assay complexity.

Inventive Principle:

Principle #6Universality (Multi-functionality)

2Measurement precision

If solid-phase immobilization is used, then detection sensitivity can be improved, but device complexity and workflow steps increase

Engineering Contradiction:

Improvedetection sensitivityVSAvoidworkflow steps

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The patent extracts the solid-phase immobilization step from the workflow, performing all reactions in liquid phase within a single container. This eliminates the complexity of solid-phase handling while maintaining detection sensitivity through the use of fluorescently labeled detectors that can be detected directly in solution.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent uses liquid phase reactions throughout the assay, utilizing fluid dynamics to mix and incubate reagents in a single container. This hydraulic approach replaces the mechanical complexity of solid-phase immobilization and transfer steps while maintaining sensitivity through homogeneous mixing.

Inventive Principle:

Principle #29Pneumatics and hydraulics

3Reliability

If nucleases are added to degrade excess detectors, then detection specificity can be improved, but assay steps and complexity increase

Engineering Contradiction:

Improvedetection specificityVSAvoidassay steps

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The patent combines the nuclease treatment step with other assay steps in the same reaction container, rather than requiring a separate step. This merging maintains detection specificity through selective degradation of excess detectors while minimizing the increase in assay complexity by integrating the step into the existing workflow.

Inventive Principle:

Principle #5Merging (Combining)

4Reliability

If detectors are designed to resist nucleases, then detection reliability can be improved, but detector design complexity increases

Engineering Contradiction:

Improvedetection reliabilityVSAvoiddetector design

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The patent applies nuclease resistance modifications selectively at specific locations on the detector molecules (such as the 5' or 3' ends), rather than throughout the entire detector structure. This local application maintains detection reliability by protecting the critical regions while minimizing detector design complexity through targeted modifications.

Inventive Principle:

Principle #3Local quality

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

This approach enables sensitive and specific detection of nucleic acids across a wide range of abundances, allowing for whole-transcriptome or miRNome multiplexing, and can be performed in a single well, optimizing resource use and simplifying the assay workflow.

Implementation Method 1

The downstream and upstream detectors are contacted with the sample and allowed to hybridize to the corresponding regions of target sequence present in the sample

Methodology Applied

Scientific EffectHybridization: Chemical Bonding

Implementation Method 2

one or more nucleases are provided during steps in the method to selectively degrade unused or excess detectors, or detectors that are not specifically hybridized to target sequences

Methodology Applied

Scientific EffectNuclease degradation: Enzyme