Single-Stranded Nucleic Acid Cleavage for Real-Time SNP Detection
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Solution Overview
Problem
Conventional methods for detecting genetic variations such as SNPs and point mutations are cumbersome, prone to cross-contamination, and require high-cost equipment and complex procedures, lacking the necessary precision and sensitivity for rapid diagnosis.
Innovation Solution
A single-stranded nucleic acid with a structure of X-Y-Z, capable of complementary bonding to a target gene, and equipped with detectable markers, is used to detect genetic variations through a cleavage reaction, eliminating the need for separate probes and enabling precise, real-time identification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiple primers or probes are used in multiplex PCR to detect different DNA targets simultaneously, then the productivity is improved, but the device complexity increases and cross reactions occur reducing measurement precision
Solution Approach 1:
The invention uses a single universal primer pair that can amplify multiple different DNA targets simultaneously through a common amplification region. Each probe contains target-specific sequences that hybridize to different DNA targets, while all probes share the same universal primer binding sites, enabling one reaction to detect multiple genetic variations without requiring multiple separate reactions
Solution Approach 2:
The invention combines multiple detection functions into a single reaction tube by using a universal primer pair alongside multiple probes with different fluorescent labels. This merging approach allows simultaneous detection of multiple DNA targets and their genetic variations in one reaction, improving productivity while managing complexity through standardized probe design
2Productivity
If multiple primers or probes are used in multiplex PCR to detect different DNA targets simultaneously, then the productivity is improved, but the measurement precision deteriorates due to cross reactions
Solution Approach 1:
The invention applies local quality by designing probes with specific fluorescent labels at different positions (5' end, 3' end, or internal) that emit distinct signals. Each probe is tailored with local modifications including fluorescent markers and quencher positions optimized for specific detection purposes, allowing multiple targets to be distinguished by their unique signal characteristics rather than requiring physically separate reactions
Solution Approach 2:
The invention uses different fluorescent labels (e.g., FAM, HEX, Cy5) that emit different colors or wavelengths of light to distinguish between multiple DNA targets and their genetic variations. This color-based differentiation allows simultaneous detection of multiple targets in one reaction without cross-contamination, maintaining measurement precision while improving productivity
3Manufacturing precision
If conventional PCR with fluorescent probes and quenchers is used to amplify specific DNA regions, then the manufacturing precision is improved, but the productivity deteriorates due to separate reactions for each target
Solution Approach 1:
The invention creates a universal amplification system where a single primer pair can amplify multiple different DNA targets simultaneously. Each probe contains a universal binding region that pairs with the universal primers, allowing the same amplification machinery to work on multiple targets at once, thereby improving productivity while maintaining the precision of specific amplification through probe-target hybridization
4Measurement precision
If complex analytical procedures and multiple reactions are used to detect genetic variations, then the measurement precision is improved, but the loss of time increases
Solution Approach 1:
The invention merges multiple detection steps into a single real-time PCR reaction. By using probes with different fluorescent labels that can be detected simultaneously by the real-time PCR system, the invention eliminates the need for separate reactions and post-PCR analysis steps, reducing detection time while maintaining precision through real-time monitoring of multiple targets
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 allows for fast and accurate discrimination of genetic variations, enhancing sensitivity and specificity in detecting mutations like KRAS and EGFR, facilitating disease diagnosis and therapeutic drug selection.
Implementation Method 1
a nucleotide sequence that can form a complementary bond to all or part of the nucleotide sequence of a single target gene containing a genetic variation
Implementation Method 2
The Y region is an RNA having a genetic sequence of one or two nucleotides at a locus of the single target gene
Data Source
AI summary
The present disclosure relates to a single nucleic acid for real-time detection for single nucleotide polymorphism (SNP) analysis of apolipoprotein E (ApoE) gene and a detection method using the same.