Fluorescent Oligonucleotide Probes for ALK Mutation Detection
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Solution Overview
Problem
Current methods for detecting ALK gene mutations, such as direct sequencing and melting curve analysis, are laborious, costly, and difficult to automate, and require specific sequence matching for fluorescent dye-labeled probes, limiting their applicability and sensitivity in predicting the efficacy of ALK inhibitors like crizotinib.
Innovation Solution
Development of fluorescently labeled oligonucleotide probes specifically designed to detect ALK(L1196M) and ALK(C1156Y) mutations using a mutation detection probe that emits different fluorescence intensities when hybridized or not hybridized to target sequences, enabling easy detection through melting curve analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If direct sequencing method is used to detect ALK gene mutations, then detection accuracy is improved, but detection complexity and cost increase significantly
Solution Approach 1:
The invention extracts only the critical mutation detection function from the complex direct sequencing process. By designing specific probes that target known mutation sites (ALK L1196M, ALK C1156Y, etc.), the method isolates the essential detection capability while eliminating unnecessary sequencing steps, thereby reducing complexity while maintaining accuracy for clinically relevant mutations.
Solution Approach 2:
The invention uses fluorescently labeled probes as simplified copies or representations of the target mutation sequences. Instead of sequencing the entire gene, specific probe sequences complementary to mutation sites are designed and used to detect the presence of mutations through hybridization, providing an accurate yet streamlined approach to mutation detection.
2Speed
If fluorescent dye-labeled nucleic acid probes are used for melting curve analysis, then detection speed is improved, but applicability is limited by sequence matching requirements
Solution Approach 1:
The invention creates a universal probe system that can detect multiple different ALK mutations (L1196M, C1156Y, and other resistance mutations) using a common fluorescent melting curve analysis platform. By designing probes with universal binding regions and variable detection regions, the system achieves broad applicability across different mutation types while maintaining fast detection speed through automated temperature cycling and fluorescence measurement.
3Productivity
If multiple nucleic acid sequences are examined simultaneously, then productivity is improved, but automation difficulty increases
Solution Approach 1:
The invention segments the detection process into distinct modular components: separate fluorescent probes for different mutations, distinct fluorescence channels for detecting different probes, and sequential temperature cycling steps. This segmentation allows multiple mutations to be examined simultaneously in a single reaction while maintaining automation through programmable temperature control and automated fluorescence detection, thereby increasing productivity without compromising automatability.
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 probes allow for sensitive and automated detection of ALK gene mutations, facilitating the evaluation of drug efficacy and tolerance, particularly for ALK inhibitor-based treatments, by simplifying the detection process and improving sensitivity.
Implementation Method 1
a mutation detection probe including at least one fluorescently labeled oligonucleotide... a fluorescence intensity of the fluorescently labeled oligonucleotide when hybridized to the target sequence is decreased or increased as compared to when not hybridized to the target sequence
Implementation Method 2
after amplifying a mutation-containing region by a PCR method, a melting curve analysis is performed using a nucleic acid probe labeled with a fluorescent dye so as to analyze a mutation in a base sequence
Implementation Method 3
melting curve analysis is performed... dissociating the hybrid by changing a temperature of the sample containing the hybrid so as to measure the change in a fluorescence signal caused by dissociation of the hybrid
Data Source
AI summary
The present invention provides a probe for detecting a mutation in the ALK gene, which is at least one fluorescently labeled oligonucleotide selected from the group consisting of P1 to P4, P7 and P8 oligonucleotides; an application thereof; and an oligonucleotide for the application.