Isothermal Nucleic Acid Detection Using Leak Absorption Oligonucleotides

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Solution Overview

Problem

Isothermal nucleic acid amplification methods face challenges with uncontrolled background signal leading to false positive results due to non-specific amplification, even in the absence of the target biomarker, which limits their effectiveness in detection strategies.

Innovation Solution

A method involving a mixture of specific oligonucleotides, including an amplification oligonucleotide, a leak absorption oligonucleotide, and a target-specific conversion oligonucleotide, along with enzymes like polymerases and nicking enzymes, is used to stabilize the non-amplified state and initiate amplification only above a predetermined threshold, thereby eliminating background amplification.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If isothermal exponential amplification is used to achieve sensitive detection of biomarkers, then amplification signal is enhanced, but background amplification occurs leading to false positive results

Engineering Contradiction:

Improvedetection sensitivityVSAvoidfalse positive rate

Core Design Contradiction:

Measurement precisionVSReliability

Solution Approach 1:

The amplification system is segmented into multiple functional oligonucleotide components (amplification oligonucleotide, leak absorption oligonucleotide, target-specific conversion oligonucleotide) that work together to enable controlled amplification only when target is present, separating the amplification function from background noise generation

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The leak absorption oligonucleotide acts as an intermediary that captures and neutralizes stray primers and non-specific amplification products, preventing them from generating false positive signals while allowing specific target amplification to proceed

Inventive Principle:

Principle #24Intermediary (Mediator)

2Power

If amplification oligonucleotides are added to enable exponential amplification, then signal amplification is achieved, but non-specific amplification of background signal occurs

Engineering Contradiction:

Improveamplification signal strengthVSAvoidnon-specific amplification

Core Design Contradiction:

PowerVSObject-generated harmful factors

Solution Approach 1:

The leak absorption oligonucleotide performs preliminary anti-action by preemptively binding and neutralizing stray primers and non-specific amplification products before they can generate harmful background signals, while allowing specific target amplification to proceed unimpeded

Inventive Principle:

Principle #9Preliminary anti-action

Solution Approach 2:

The system changes the kinetic parameters of the amplification reaction by introducing threshold-dependent activation, where amplification only proceeds when target concentration exceeds a predetermined threshold, fundamentally altering the amplification profile from continuous to switch-like behavior

Inventive Principle:

Principle #35Parameter changes

3Measurement precision

If the amplification system is made highly sensitive to detect low copy number targets, then detection limit is improved, but background amplification becomes more prominent

Engineering Contradiction:

Improvedetection limitVSAvoidbackground signal

Core Design Contradiction:

Measurement precisionVSObject-affected harmful factors

Solution Approach 1:

The system implements positive feedback through the target-specific conversion oligonucleotide that generates additional amplification oligonucleotides when target is present, creating a self-amplifying loop that only activates when target concentration exceeds the threshold, while the leak absorption oligonucleotide provides negative feedback by continuously removing stray primers

Inventive Principle:

Principle #23Feedback

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 effectively eliminates background amplification, enabling robust, ultra-sensitive, and ultra-specific detection of nucleic acid targets by ensuring that amplification occurs only when the target is present, thus improving the reliability of detection methods.

Implementation Method 1

a first, a second and a third oligonucleotides into the mixture, wherein the first oligonucleotide is an amplification oligonucleotide, the second oligonucleotide is a leak absorption oligonucleotide

Methodology Applied

Scientific EffectNicking enzyme activity: Enzyme

Implementation Method 2

enzymes like polymerases and nicking enzymes, is used to stabilize the non-amplified state and initiate amplification only above a predetermined threshold

Methodology Applied

Scientific EffectPolymerase activity: Enzyme

Implementation Method 3

Method of eliminating background amplification in isothermal amplification of nucleic acid targets

Methodology Applied

Scientific EffectIsothermal amplification:

Implementation Method 4

isothermal exponential amplification of the nucleic acid target

Methodology Applied

Scientific EffectDNA polymerization:

Data Source

PatentUS11111529B2Method of eliminating background amplification of nucleic acid targets

Publication Date: 2021.09.07 CENT NAT DE LA RECH SCI (C N R S)

AI summary

An object is to provide a method of eliminating background amplification in detection of rare nucleic acids based on isothermal amplification. The method comprising steps of: preparing a mixture including solution and enzymes, and adding a first and a second template into the mixture, wherein the first template is an amplification template, and the second template is a leak absorption template. Methods to use this approach for multiplexed, ultrasensitive and ultra-specific detection of nucleic acids are presented.