Oligonucleotide Probe Pair for High-Throughput Nucleic Acid Detection
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Solution Overview
Problem
Current gene mutation detection and nucleic acid sequencing methods are cumbersome, costly, and often produce false positives due to non-specific hybridization, requiring complex processes and labor-intensive multi-color fluorescence systems, which limits their scalability and accuracy.
Innovation Solution
A novel detection probe and oligonucleotide chip system using a detection probe pair (P1 and P2) that anneals to a nucleic acid sample, with a gap filled by complementary deoxyribose nucleotides in the presence of DNA polymerase and ligase, allowing for ligation and subsequent amplification, using universal primers and a single-color fluorescence label for high-throughput, low-cost, and high-specificity analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional nucleic acid hybridization methods are used, then high hybridization specificity and detection sensitivity are achieved, but the process becomes complicated with too many operation steps and radioactive labeling that damages human bodies
Solution Approach 1:
The patent extracts and eliminates the radioactive labeling step from the traditional nucleic acid hybridization process, replacing it with non-radioactive detection methods. This removes the harmful element while maintaining the core hybridization function, thereby reducing process complexity and safety concerns while preserving detection sensitivity and specificity.
Solution Approach 2:
The patent employs disposable, non-radioactive probes that can be synthesized cheaply and used for single or limited applications. These probes replace expensive, hazardous radioactive labels, reducing both process complexity and safety risks while maintaining adequate detection performance for the intended application scope.
2Measurement precision
If PCR-based detection technologies are used, then sensitivity and specificity are greatly enhanced, but the methods cannot identify the type of mutation and can only detect a part of SNP
Solution Approach 1:
The patent designs a universal detection system using conserved primer binding sites that can detect multiple types of mutations and SNPs simultaneously. The probes are engineered with universal regions for amplification and specific regions for mutation detection, enabling a single system to handle diverse genetic variations, thereby improving both detection coverage and maintaining high sensitivity through PCR amplification.
3Measurement precision
If gel electrophoresis-based sequencing technology is used, then accurate identification of mutation location and type is achieved, but the process becomes time-consuming
Solution Approach 1:
The patent replaces the mechanical gel electrophoresis separation system with a hybridization-based detection system using specific probes. Instead of physically separating DNA fragments by size through electrophoresis, the method uses complementary base pairing between probes and target sequences to directly identify mutations, dramatically reducing detection time while maintaining accurate identification of mutation location and type.
4Adaptability or versatility
If multi-color fluorescence systems are used, then comprehensive analysis is achieved, but costs increase and the analysis process becomes complex
Solution Approach 1:
The patent employs universal primers and probes with conserved sequences that can detect multiple mutation types using a single fluorescence labeling system. Instead of requiring different fluorescent labels for different mutations, the universal design allows one color system to provide comprehensive analysis through specific probe-target hybridization patterns, thereby reducing system complexity while maintaining analytical comprehensiveness.
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 costs, enhances specificity and sensitivity, and facilitates high-throughput detection by using universal primers and a single-color fluorescence label, simplifying the analysis process and eliminating the need for complex probe optimization and multiplex PCR, thereby improving the practicality of gene mutation and sequencing analysis.
Implementation Method 1
The detection probe pair P1 and P2 anneals and hybridizes to a nucleic acid sample to be detected
Implementation Method 2
the base complementary to that at the base site to be detected will fill the gap in the presence of DNA polymerase and DNA ligase
Implementation Method 3
the base complementary to that at the base site to be detected will fill the gap in the presence of DNA polymerase and DNA ligase, and thus the detection probe pair P1 and P2 is ligated into one single probe
Data Source
AI summary
High-throughput detection for the interesting base or the mutation site in the nucleic acid sample can be achieved by means of the linear test probe pairs P1 and P2. The test probe pairs P1 and P2 respectively comprise either of the flanking complementary sequences which are adjacent to the interesting base or the mutation site in the nucleic acid sample. The invention can be applied to the re-sequencing the target nucleic acid sequence, the detection and analysis for the mutation, insertion, or deletion sites of a known nucleic acid sequence, and the genotyping of the pathogenic microorganism.