Nucleic Acid Detection Using Single-Stranded Tag Primers
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Solution Overview
Problem
Current nucleic acid detection methods are costly, time-consuming, and require specialized equipment, making them inefficient for genetic diagnosis and testing, particularly in clinical settings where they burden patients and testers with lengthy procedures and expensive equipment needs.
Innovation Solution
A method using primers with a tag region linked to the 5' end that remains single-stranded during PCR, allowing for the amplification of double-stranded DNA with single-stranded regions at each end, which can be detected using oligonucleotide probes without the need for special equipment, enabling simple and accurate visual observation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If heat treatment is used to denature double-stranded nucleic acid for hybridization, then hybridization can be achieved, but the process becomes troublesome and hybridization efficiency is reduced due to reannealing
Solution Approach 1:
The PCR primers are pre-designed with single-stranded tag regions that remain unpaired after amplification. This preliminary structural arrangement eliminates the need for subsequent heat denaturation steps, as the single-stranded regions are already available for immediate hybridization with probes.
Solution Approach 2:
The invention extracts and utilizes only the necessary single-stranded regions from the amplified DNA product by incorporating them as tag regions in the primers. This allows hybridization to occur without requiring separation of the entire double-stranded molecule through heat treatment.
2Measurement precision
If fluorescent labeling is used to detect target nucleic acid, then detection specificity is enhanced, but the cost increases significantly
Solution Approach 1:
The invention replaces expensive fluorescent labels with inexpensive oligonucleotide probes that can be synthesized at low cost. These probes provide sufficient detection capability without requiring expensive labeling reagents or specialized detection equipment.
Solution Approach 2:
The invention uses sequence-complementary oligonucleotide probes as informational copies of the target region. These probes hybridize specifically to the target sequence, providing detection capability through base-pairing recognition rather than through expensive fluorescent labeling.
3Adaptability or versatility
If multiple antigen-antibody or ligand-receptor pairs are used for labeling, then multiple target nucleic acids can be detected, but the number of usable combinations is limited
Solution Approach 1:
The invention creates a universal detection system based on oligonucleotide hybridization that can detect any target sequence. By using sequence-complementary probes rather than target-specific antibodies or ligands, the system achieves broad versatility without being limited by the availability of specific biological reagents.
Solution Approach 2:
The invention changes the detection parameter from relying on specific antigen-antibody or ligand-receptor interactions to relying on sequence-complementary hybridization. This parameter change enables unlimited multiplexing capability, as probes can be designed for any nucleic acid sequence without being constrained by biological reagent availability.
4Measurement precision
If single-stranded DNA is used for detection, then hybridization can occur, but the DNA tends to curl into balls and detection sensitivity is reduced
Solution Approach 1:
The double-stranded amplified DNA product serves as an intermediary structure that maintains structural stability. The single-stranded tag regions extend from this stable double-stranded backbone, preventing the entire molecule from curling into balls while still providing accessible single-stranded regions for probe hybridization.
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 reduces the complexity and cost of nucleic acid detection by allowing for rapid, specific, and low-cost analysis of multiple targets without requiring expensive labeling or specialized equipment, improving detection sensitivity and efficiency.
Implementation Method 1
allowing said double-stranded DNA having a single-stranded region at each end to hybridize with a first oligonucleotide probe immobilized on a zone different from the application zone on the chromatographic carrier
Data Source
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AI summary
An object of the present invention is to provide methods for amplifying and detecting a nucleic acid that allow efficient hybridization, and devices and kits for use in the methods. The present invention includes amplifying a target nucleic acid into a double-stranded nucleic acid having a single-stranded region at each end, and detecting this nucleic acid. The present invention also provides detection devices and kits that make use of these methods.