Plasmid Vector Self-Amplification for Sensitive Gene Detection

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Solution Overview

Problem

Current methods for detecting gene promoter activity, especially at early stages of diseases, face challenges due to low sensitivity and safety concerns with virus-based reporter vectors, while plasmid vectors lack sufficient amplification capabilities for enhanced detection.

Innovation Solution

A plasmid vector design that includes a gene promoter, a reporter gene, an internal ribosome entry site, a replication origin-binding protein gene, and a replication origin sequence, allowing self-amplification in activated host cells to enhance detection sensitivity.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If a virus reporter vector is used to increase detection sensitivity through viral proliferation, then the sensitivity of gene promoter activity detection is improved, but safety risks and handling convenience deteriorate due to infection hazards

Engineering Contradiction:

Improvedetection sensitivityVSAvoidinfection risk

Core Design Contradiction:

Measurement precisionVSObject-affected harmful factors

Solution Approach 1:

The patent extracts the replication capability from the virus vector and transfers it to the plasmid vector by incorporating a replication origin sequence. This allows the plasmid to self-amplify in host cells without requiring viral infection, thereby eliminating infection risks while maintaining the amplification function needed for high-sensitivity detection

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The plasmid vector system creates multiple copies of the reporter gene through plasmid replication rather than viral proliferation. The replication origin sequence enables the plasmid to be copied repeatedly in host cells, generating sufficient reporter protein signal for sensitive detection without the harmful effects of viral infection

Inventive Principle:

Principle #26Copying

2Ease of operation

If a plasmid reporter vector is used to improve safety and handling convenience, then safety and ease of operation are improved, but detection sensitivity deteriorates due to lack of amplification capability

Engineering Contradiction:

Improvehandling convenienceVSAvoiddetection sensitivity

Core Design Contradiction:

Ease of operationVSMeasurement precision

Solution Approach 1:

The plasmid vector is pre-equipped with a replication origin sequence and origin-binding protein gene before introduction into host cells. This preliminary configuration enables the plasmid to autonomously replicate and amplify the reporter gene signal within the cells, ensuring high detection sensitivity without requiring complex viral infection procedures

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The plasmid vector achieves self-amplification through its own replication origin sequence and the host cell's replication machinery. The system serves itself by utilizing cellular resources to replicate the plasmid and produce reporter protein, eliminating the need for viral vectors while maintaining amplification capability for sensitive detection

Inventive Principle:

Principle #25Self-service

3Quantity of substance

If the amount of reporter protein expression is increased through viral proliferation to enhance detection sensitivity, then detection sensitivity is improved, but the complexity of the system increases due to viral culture requirements

Engineering Contradiction:

Improvereporter protein amountVSAvoidsystem complexity

Core Design Contradiction:

Quantity of substanceVSDevice complexity

Solution Approach 1:

The patent changes the fundamental parameter of the vector system from viral to plasmid-based replication. By incorporating a replication origin sequence compatible with host cell machinery, the system achieves reporter protein amplification through plasmid replication rather than viral proliferation, significantly reducing system complexity while maintaining high protein expression levels for sensitive detection

Inventive Principle:

Principle #35Parameter changes

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

The plasmid vector significantly increases the sensitivity of gene promoter activity detection by amplifying reporter protein expression, offering a safer and more convenient alternative to virus-based systems.

Implementation Method 1

an internal ribosome entry site which links the reporter gene to the replication origin-binding protein gene

Methodology Applied

Scientific EffectInternal ribosome entry site (IRES) mechanism:

Implementation Method 2

a replication origin sequence which is recognized by the replication origin-binding protein; allowing self-amplification in activated host cells

Methodology Applied

Scientific EffectDNA replication:

Data Source

PatentUS9783810B2Plasmid vector, method for detecting gene promoter activity, and assay kit

Publication Date: 2017.10.10 TOSHIBA MEDICAL SYST CORP

AI summary

According to one embodiment, a first gene encodes a reporter protein. The first gene is disposed at the downstream of the gene promoter. A second gene is disposed at the downstream of the gene promoter and encodes a replication origin-binding protein. An internal ribosome entry site is disposed between the first gene and the second gene. The transcription termination signal sequence encodes a signal for terminating the transcription of the first gene and the second gene. A replication origin sequence is recognized by the replication origin-binding protein.