Oligonucleotide Probes for PI3KCA Mutation Detection
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Solution Overview
Problem
Current methods for detecting somatic mutations in the PI3KCA gene, particularly in cancer diagnostics, face challenges in precision and specificity, especially when multiple mutations are present, which affects the effectiveness of targeted cancer therapies.
Innovation Solution
The development of oligonucleotides that are at least 90% identical to a specific sequence with 3 or fewer mismatches at the 3'-terminus, including modified nucleotides, for detecting mutations such as H1047Y and others in the PI3KCA gene, allowing for precise discrimination and simultaneous detection of multiple mutations using allele-specific PCR.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If allele-specific primers are used for detecting PI3KCA mutations, then detection capability is provided, but specificity and precision are insufficient when multiple mutations are present
Solution Approach 1:
The patent applies local quality by introducing modified nucleotides (such as 2'-O-methyl, LNA, or phosphorothioate modifications) specifically at the 3'-terminal positions of the oligonucleotide probes. These local modifications enhance the discrimination capability between wild-type and mutant sequences without affecting the overall probe structure or requiring complete redesign of all probes in the panel.
Solution Approach 2:
The patent changes the chemical parameters of the oligonucleotide probes by incorporating modified nucleotides with different binding affinities and thermal stability characteristics. This allows for enhanced specificity in distinguishing single-nucleotide differences while maintaining the ability to detect multiple mutations simultaneously through adjusted annealing temperatures and hybridization conditions.
2Quantity of substance
If multiple mutations are detected simultaneously, then comprehensive diagnostic information is obtained, but discrimination accuracy between different mutations decreases
Solution Approach 1:
The patent segments the detection process by designing separate oligonucleotide probes for different mutation types (exon 9 mutations, exon 20 mutations, and other mutations) that can be used in combination. Each probe is optimized with specific modified nucleotides at its 3'-terminal region to maintain high discrimination accuracy for its target mutation while allowing multiplexed detection of multiple mutations in the same sample.
Solution Approach 2:
The patent applies preliminary action by pre-optimizing the oligonucleotide probes with specific modified nucleotides and determining their optimal concentrations and annealing temperatures before clinical use. This pre-characterization allows for accurate discrimination of multiple mutations simultaneously without requiring complex real-time optimization during patient testing.
3Ease of manufacture
If standard oligonucleotides are used for mutation detection, then simplicity is maintained, but precision and reliability of detection are reduced
Solution Approach 1:
The patent uses composite materials by combining standard nucleotides with modified nucleotides in the oligonucleotide probes. The probes consist of a standard nucleotide sequence for target recognition and specificity, with modified nucleotides (such as 2'-O-methyl, LNA, or phosphorothioate) incorporated at strategic positions to enhance binding affinity and discrimination capability, thereby improving detection reliability while maintaining ease of synthesis through commercial availability of modified nucleotide building blocks.
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 enables rapid and precise determination of PI3KCA mutations, improving the accuracy of cancer diagnosis and predicting treatment effectiveness by enhancing allelic discrimination and multiplexing capabilities.
Implementation Method 1
Oligonucleotides for detecting a mutation H1047Y in the human PIK3CA gene, that are at least 90% identical to and have the 3'-terminal nucleotide of SEQ ID NO: 39
Implementation Method 2
allele-specific PCR
Data Source
AI summary
The invention comprises reagents and methods for detecting cancer-associated mutations in the human PI3KCA (PIK3CA) gene and assessing the patients based thereon.