Sensor DNA Composition for Short Nucleic Acid Detection
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Solution Overview
Problem
Current nucleic acid detection methods, such as PCR and isothermal amplification, face limitations in detecting short targets and require expensive thermal cycling instruments, and traditional detection methods are limited to double-stranded DNA binding dyes or probe sequences, lacking versatility in detecting transcription or translation products.
Innovation Solution
A composition comprising sensor DNA with a target binding region and a reporter gene linked to a promoter, allowing expression of a reporter nucleic acid or protein only in the presence of the target nucleic acid, utilizing cell-free replication, transcription, and protein synthesis to detect nucleic acids through signal generation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If PCR-based testing is used to amplify target nucleic acids, then detection sensitivity is improved, but expensive thermal cycling instruments are required
Solution Approach 1:
The invention extracts the essential function of nucleic acid amplification from the complex thermal cycling instrument requirement. By using isothermal amplification methods (such as LAMP, RPA, or NASBA) that operate at constant temperatures, the patent eliminates the need for expensive thermal cyclers while maintaining amplification capability. The sensor DNA construct is designed to work specifically under isothermal conditions, separating the amplification function from the complex temperature control machinery.
Solution Approach 2:
The invention changes the operating temperature parameter from dynamic thermal cycling to static isothermal conditions. The sensor DNA is engineered with specific melting temperatures and binding characteristics that function optimally at constant temperatures (e.g., 60-65°C for LAMP, 37-42°C for RPA). This parameter change fundamentally simplifies the instrumentation requirements while preserving detection sensitivity.
2Device complexity
If LAMP reaction is used for isothermal amplification, then expensive instruments are eliminated, but detection of short targets is limited
Solution Approach 1:
The sensor DNA construct is designed with universal applicability across different target lengths and types. The modular structure consists of a generic promoter region, a customizable target binding region, and a reporter gene. By adjusting only the target binding region sequence while keeping the functional architecture constant, the system can detect various target lengths including short targets, making the method universally applicable beyond LAMP's traditional limitations.
Solution Approach 2:
The sensor DNA is segmented into distinct functional modules: a promoter region that drives transcription, a target binding region that provides sequence-specific recognition, and a reporter gene that generates the detection signal. This segmentation allows independent optimization of each module - the target binding region can be designed for short targets while the promoter and reporter components remain unchanged, overcoming the length limitation of traditional LAMP.
3Measurement precision
If traditional detection methods using double-stranded DNA binding dyes or probe sequences are used, then amplification detection is achieved, but versatility in detecting transcription or translation products is limited
Solution Approach 1:
The invention introduces an intermediary transcription-translation system between the target nucleic acid and the detection signal. The sensor DNA acts as a template that, when bound to the target, drives transcription of mRNA and subsequent translation into reporter proteins. This intermediary biological system serves as a universal translator that can detect not only nucleic acids but also verify functional expression, bridging the gap between genetic material and phenotypic expression detection.
Solution Approach 2:
The invention replaces the mechanical/chemical detection system (DNA binding dyes or fluorescent probes) with a biological expression system. Instead of directly detecting nucleic acids through physical-chemical interactions, the system uses the cell's transcription and translation machinery to convert target binding into protein synthesis. This substitution enables detection of functional expression and extends versatility to detect transcriptional and translational products.
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 simple and precise detection of nucleic acids by measuring reporter gene expression, applicable in various fields including molecular diagnosis, and adaptable to different target nucleic acids and environments.
Implementation Method 1
a target binding region annealing to a target sequence
Implementation Method 2
target binding region annealing to a target sequence
Implementation Method 3
cell-free replication, transcription, and protein synthesis
Implementation Method 4
cell-free replication, transcription, and protein synthesis
Implementation Method 5
signal generation through expression of the reporter nucleic acid or reporter protein
Implementation Method 6
signal generation through expression of the reporter nucleic acid or reporter protein
Data Source
AI summary
The present invention relates to a composition for detecting a target nucleic acid sequence and a method of detecting a nucleic acid using the same.