Isothermal Nucleic Acid Amplification via Nicking Enzyme and Polymerase

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Solution Overview

Problem

Current nucleic acid amplification techniques, such as PCR and isothermal methods, require temperature cycling, specialized equipment, and often involve complex processes, making them inefficient for rapid and specific amplification of short DNA or RNA sequences.

Innovation Solution

A method using only two templates and one or two nicking enzymes, along with a thermophilic polymerase, under isothermal conditions, which eliminates the need for initial heat denaturation and allows for rapid amplification of 20-30mer products up to 1010-fold in 2.5 to 10 minutes without specialized equipment.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If PCR temperature cycling is used for nucleic acid amplification, then amplification specificity and reliability are improved, but reaction time and power consumption increase

Engineering Contradiction:

Improveamplification specificityVSAvoidreaction time

Core Design Contradiction:

ReliabilityVSLoss of time

Solution Approach 1:

The patent changes the temperature parameter from cycling (PCR) to constant isothermal conditions (37-65°C), eliminating the time-consuming temperature cycling steps while maintaining amplification through the coordinated action of nicking enzymes and polymerases at a single optimized temperature

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent implements continuous exponential amplification at constant temperature without interruption for temperature cycling, where nicking enzymes continuously generate nicks and polymerases continuously extend primers, creating an uninterrupted amplification process that reduces reaction time from 20-30 minutes to under 10 minutes

Inventive Principle:

Principle #20Continuity of useful action

2Loss of time

If isothermal amplification methods are used to eliminate temperature cycling, then reaction time and equipment requirements are reduced, but amplification robustness and sensitivity decrease

Engineering Contradiction:

Improvereaction timeVSAvoidamplification robustness

Core Design Contradiction:

Loss of timeVSReliability

Solution Approach 1:

The patent merges the functions of multiple enzymes (nicking enzymes and polymerases) working cooperatively at isothermal conditions, where nicking enzymes create nicks that generate new primer binding sites for polymerases, which then extend to create more nicking sites, forming a synergistic amplification cycle that maintains robustness without temperature cycling

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The patent introduces nicking enzymes as intermediary agents that convert double-stranded DNA into single-stranded regions with nicks, serving as intermediates that enable primer binding and polymerase extension at isothermal conditions, thereby bridging the gap between template DNA and amplification products without requiring thermal denaturation

Inventive Principle:

Principle #24Intermediary (Mediator)

3Measurement precision

If complex amplification processes with multiple steps are used, then amplification sensitivity is improved, but process complexity and equipment requirements increase

Engineering Contradiction:

Improvedetection sensitivityVSAvoidprocess complexity

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The patent extracts and eliminates the complex temperature cycling step from the amplification process, retaining only the essential isothermal nicking and extension steps that can be performed in a single reaction tube at constant temperature, thereby reducing process complexity while maintaining detection sensitivity through exponential amplification

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent implements a self-sustaining amplification cycle where nicking enzymes and polymerases work autonomously at isothermal conditions, with nicking creating substrates for polymerase extension, and extension products creating new nicking sites, eliminating the need for external temperature control equipment and complex procedural interventions

Inventive Principle:

Principle #25Self-service

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 method enables quick and efficient amplification of nucleic acid sequences, reducing reaction time and equipment requirements while maintaining high specificity and sensitivity, suitable for detecting low copy numbers of target sequences.

Implementation Method 1

a nicking enzyme binding to the template nucleic acid and a nicking site at which the nicking enzyme is capable of nicking the template nucleic acid

Methodology Applied

Scientific EffectEnzymatic cleavage: Enzyme

Implementation Method 2

a polymerase capable of extending a primer along a template

Methodology Applied

Scientific EffectNucleic acid polymerization:

Implementation Method 3

a stabilizing region 5′ of the nicking enzyme binding site and the nicking site

Methodology Applied

Scientific EffectThermal stabilization:

Data Source

PatentUS20240409987A1Nicking and extension amplification reaction for the exponential amplification of nucleic acids

Publication Date: 2024.12.12 IONIAN TECHNOLOGIES LLC

AI summary

The invention is in general directed to the rapid exponential amplification of short DNA or RNA sequences at a constant temperature.