Probe and Primer Library for Rapid ctDNA Mutation Detection
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Solution Overview
Problem
Existing methods for detecting cancer-related gene mutations in ctDNA are limited in their applicability to a wide range of cancers and require separate identification of patient-specific mutations, making them inefficient for immediate and comprehensive cancer relapse detection.
Innovation Solution
A library of primers and probes designed for detecting frequent genetic mutations in genes such as ARID1A, BRAF, EGFR, FGFR2, FGFR3, HRAS, KRAS, and PIK3CA, optimized for digital PCR, allowing for quick and sensitive analysis of multiple cancer types under standardized conditions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If a comprehensive library of primers and probes for multiple cancer genes is prepared in advance, then the ability to detect various cancers immediately is improved, but the complexity of the library system increases
Solution Approach 1:
The library is segmented into multiple independent primer-probe sets, each targeting specific genes (ARID1A, BRAF, EGFR, FGFR2, FGFR3, HRAS, KRAS, PIK3CA). Each set can be independently selected and used for detecting specific cancer types, allowing comprehensive coverage while maintaining manageable complexity through modular organization.
Solution Approach 2:
The library creates a universal detection system that can identify mutations across multiple cancer types using a standardized format. All primer-probe sets follow consistent design principles and can be used with the same digital PCR methodology, enabling one library to serve multiple detection purposes simultaneously.
2Measurement precision
If separate identification of patient-specific mutations is performed using NGS, then detection accuracy is improved, but the time and cost for preparation increases
Solution Approach 1:
The library performs preliminary action by pre-identifying and preparing primer-probe sets for all major cancer-related gene mutations before clinical use. Common mutations in genes like KRAS, EGFR, and BRAF are anticipated and ready for immediate detection, eliminating the need for time-consuming NGS sequencing and custom primer design in routine clinical settings.
Solution Approach 2:
Instead of creating custom detection systems for each patient through NGS, the library uses standardized, pre-validated primer-probe copies that have been optimized for digital PCR. These ready-made sets replicate the detection capability needed for common mutations without requiring individual customization.
3Measurement precision
If primers and probes are optimized for each specific mutation, then detection sensitivity is improved, but the number of required primer-probe sets increases
Solution Approach 1:
The library optimizes detection sensitivity by carefully controlling parameters such as primer concentrations (450-3600 nM), annealing temperatures (55-61°C), and adding specific reagents like 7-deaza-dGTP (20-500 μM) or DMSO (0.15-3.8%). These parameter adjustments enable highly sensitive detection using a manageable number of primer-probe sets.
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
Enables immediate and early detection of cancer relapse across various cancers by providing a comprehensive set of primers and probes that can be used under uniform conditions, facilitating rapid and sensitive mutation analysis in digital PCR.
Implementation Method 1
two types of probes (mutant and wild type) that complementarily bind to parts of the amplified gene sequences
Data Source
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AI summary
An analytical means quickly applicable to more cancers is provided. A library constituted by a plurality of probes and/or primers for detecting a mutation relevant to a cancer in a gene selected from the group consisting of ARID1A, BRAF, EGFR, FGFR2, FGFR3, HRAS, KRAS, and PIK3CA or a promoter region thereof, wherein the plurality of probes and/or primers include those for detecting c.5965C>T, c.5548dup, and c.3826C>T for ARID1A; c.1799T>A for BRAF; c.2573T>G, c.2235 _2249del, and c.2236_2250del for EGFR; c.755C>G, c.1650T>A, and c.1147T>C for FGFR2; c.746C>G, c.1124A>G, and c.742C>T for FGFR3; c.37G>C, c.182A>G, c.35G>T, c.182A>T, c.181C>A, c.34G>A, and c.35G>A for HRAS; c.35G>A, c.35G>T, c.38G>A, and c.34G>T for KRAS; and c.3140A>G, c.1633G>A, and c.1624G>A for PIK3CA.