Mutant Nucleic Acid Detection via Nuclease and Fluorescent Probes
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Solution Overview
Problem
Current methods for detecting low-concentration, low-frequency mutations in nucleic acids, such as those found in cancer-related ctDNA, suffer from low sensitivity and high costs, and are difficult to multiplex, making accurate diagnosis challenging.
Innovation Solution
A method involving amplification with phosphate-group-bound and unbound primers, followed by nuclease treatment to obtain single-stranded nucleic acids, fixation to a detection chip, and use of fluorescently labeled probes to detect mutations, with additional probes to target wild-type nucleic acids, enabling efficient and multiplexed detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If next-generation sequencing (NGS) is used to detect low-frequency mutations, then comprehensive genetic mutation analysis is provided, but sensitivity is limited (LoD up to 1% VAF) and processing time is long
Solution Approach 1:
The method segments the detection process into distinct steps: amplification with asymmetric phosphorylation, selective nuclease digestion to generate single-stranded DNA, immobilization on chip surface, and probe-based detection. This segmentation allows each step to be optimized independently, achieving both comprehensive mutation analysis and high sensitivity (0.01% VAF detection limit).
Solution Approach 2:
The patent introduces several intermediary elements: biotinylated primers as intermediaries for amplification, lambda exonuclease as an intermediary to selectively digest phosphorylated strands, and fluorescent probes as intermediaries for specific mutation detection. These intermediaries enable the system to achieve high sensitivity without sacrificing comprehensive analysis capability.
2Measurement precision
If advanced approaches with unique molecular identifiers (UMIs) are used, then sensitivity is improved, but costs increase significantly
Solution Approach 1:
The patent replaces expensive UMIs with a cost-effective approach using simple fluorescent probes that bind to immobilized DNA. The probes are consumed in the detection process but are inexpensive compared to UMI synthesis and processing, achieving high sensitivity at lower cost.
Solution Approach 2:
The method extracts only the essential function of UMIs (enhancing detection sensitivity) while eliminating the costly components. By using asymmetric phosphorylation and selective nuclease digestion to generate unique single-stranded templates, the patent achieves UMI-like sensitivity improvement without the associated costs.
3Measurement precision
If digital PCR (dPCR) is used to detect low-frequency mutations, then sensitivity is improved (LoD up to 0.1% VAF), but multiplexing is almost impossible
Solution Approach 1:
The patent creates a universal detection platform where multiple fluorescent probes with different emission wavelengths can simultaneously detect multiple mutations. The immobilization-based approach and optical detection system allow multiplexing while maintaining the high sensitivity (0.01% VAF) achieved by the asymmetric amplification and nuclease digestion steps.
Solution Approach 2:
The patent transitions from the single-dimension detection of dPCR to multi-dimensional detection by using fluorescent probes with different emission spectra. This allows simultaneous detection of multiple mutations in different dimensions of the electromagnetic spectrum while maintaining high sensitivity on the same chip platform.
4Reliability
If circulating tumor DNA (ctDNA) is detected in early stage cancer, then reliable prognostic and predictive marker information is obtained, but the variant allele frequency (VAF) is extremely low and quantification is difficult due to excessive wild-type cell-free DNA
Solution Approach 1:
The patent applies local quality enhancement by making the DNA strands asymmetric through differential phosphorylation. This creates locally distinct properties (phosphorylated vs. non-phosphorylated 5' ends) that enable selective digestion and enrichment of mutant alleles, dramatically improving VAF quantification accuracy even at extremely low frequencies in early-stage cancer.
Solution Approach 2:
The method performs preliminary enrichment of mutant alleles through asymmetric amplification and selective nuclease digestion before detection. This preliminary action removes the overwhelming background of wild-type DNA, allowing accurate quantification of extremely low VAF ctDNA in early-stage cancer patients.
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 method reduces false positives and enhances detection efficiency, allowing for high-sensitivity detection of low-frequency mutations even at very low concentrations, with the ability to detect multiple mutations simultaneously.
Implementation Method 1
treating the amplified product with a nuclease that specifically binds to the phosphate group to obtain a single-stranded first target nucleic acid to which the phosphate group is not bound
Implementation Method 2
contacting it with a first probe labeled with a first fluorescent molecule
Data Source
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AI summary
The present invention relates to a method for detecting a mutant gene, wherein a nucleic acid targeting the mutant gene and its wild-type gene is used to increase detection speed and efficiency, reduce the false positive rate, and enable multiplex detection of low-concentration and low-frequency mutations.