Fluorescent Oligonucleotide Probe for EGFR Polymorphism Detection
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Solution Overview
Problem
Current methods for detecting EGFR exon 19 polymorphisms, such as PCR-RFLP and melting curve analysis, are labor-intensive and prone to contamination, making them inefficient for automated detection and requiring significant effort for analyzing multiple genetic polymorphisms.
Innovation Solution
A probe system comprising fluorescence-labeled oligonucleotides with specific sequences (SEQ ID NOs: 5, 4, and 6) is used for detecting EGFR exon 19 polymorphisms, allowing for simple and reliable determination of one-base differences, even in samples with both wild-type and mutant variants, using Tm analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If PCR-RFLP method is used for polymorphism detection, then detection accuracy is achieved, but labor intensity increases and contamination risk occurs
Solution Approach 1:
The patent replaces the mechanical/manual PCR-RFLP method with an automated fluorescent probe-based detection system. The probe hybridizes specifically to target sequences and generates fluorescent signals that can be automatically read by instrumentation, eliminating manual intervention steps while maintaining detection accuracy.
Solution Approach 2:
The patent introduces a fluorescent probe as an intermediary molecule that bridges the target DNA sequence and the detection system. The probe contains a fluorophore and quencher that generate detectable signals upon hybridization, serving as a mediator that enables automated detection without direct manual manipulation of the DNA samples.
2Measurement precision
If PCR-RFLP method is used for polymorphism detection, then detection accuracy is achieved, but automation difficulty increases
Solution Approach 1:
The patent replaces manual operations with an automated fluorescent detection system where probes hybridize to target sequences and generate fluorescent signals that can be automatically quantified and analyzed by instrumentation, enabling full automation of the detection process while preserving accuracy.
Solution Approach 2:
The fluorescent probe performs self-detection by automatically hybridizing to complementary target sequences and generating fluorescent signals without requiring manual intervention. The system is self-contained and can be automated, with the probe itself serving as both the reagent and the detection element.
3Difficulty of detecting and measuring
If melting curve analysis is used for polymorphism detection, then detection capability is achieved, but contamination risk increases
Solution Approach 1:
The patent uses a fluorescent probe as an intermediary that specifically binds to the target sequence and generates signals only from the intended target, preventing contamination from other DNA sources. The probe's specific hybridization ensures that only complementary sequences produce signals, eliminating false positives from contamination.
Solution Approach 2:
The patent replaces the open melting curve analysis system with a closed fluorescent probe-based system where signals are generated within the reaction mixture itself, eliminating the need for post-PCR handling and reducing contamination risk from external sources.
4Adaptability or versatility
If multiple polymorphisms are analyzed using conventional methods, then comprehensive detection is achieved, but time consumption increases
Solution Approach 1:
The patent combines multiple polymorphism detection capabilities into a single fluorescent probe assay. Multiple probes can be used simultaneously in one reaction mixture, each detecting different polymorphisms, thereby consolidating multiple detection steps into one unified process that reduces time while maintaining comprehensive coverage.
Solution Approach 2:
The fluorescent probe system provides universal detection capability that can identify multiple different polymorphisms using the same basic assay platform. By designing probes with different fluorescent labels or using a single probe that detects multiple variants, the system achieves multi-functionality without requiring separate assays for each polymorphism.
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 probe system enables efficient and reliable detection of EGFR exon 19 polymorphisms, facilitating the selection of appropriate cancer treatments by determining the efficacy or resistance to EGFR-TKI therapies, and can be applied in both medical and biochemical contexts.
Implementation Method 1
a hybrid (double-stranded DNA) is formed from target single-stranded DNA from a detection sample and a probe that is complementary to the sequence of interest including the gene polymorphism that is the target of detection
Implementation Method 2
The dissociation (melting) of the hybrid resulting from the temperature increase is detected by signal measurement using absorbance or the like to thereby determine the presence or absence of a target polymorphism by determining a Tm value
Implementation Method 3
the cytosine at the 5' terminus is fluorescence-labeled
Data Source
Figure 1(A)~3(D)
Figure 4(A)~6(C)
Figure 7(A)~8(D)
AI summary
A polymorphism-detecting probe, an amplification primer and the use thereof are provided to enable simple and highly reliable determination of different polymorphisms in an EGFR gene. A fluorescence-labeled oligonucleotide selected from at least one of P5 - P7 below is used as the polymorphism-detecting probe. (P5) An oligonucleotide comprising or consisting of a sequence having homology with a nucleotide sequence having a length of 9 - 50 bases comprising base positions 104 - 112 of the base sequence of SEQ ID NO: 2, and in which the base that is homologous to base number 112 is thymine, the base that is homologous to base number 104 is cytosine, and the cytosine is fluorescence-labeled; (P6) An oligonucleotide comprising or consisting of a sequence having homology with a nucleotide sequence having a length of 16 - 50 bases comprising base positions 104 - 119 of the base sequence of SEQ ID NO: 2, and in which the base at position 119 is substituted by a base other than guanine, and in which the base that is homologous to base number 104 is cytosine, and the cytosine is fluorescence-labeled; and (P7) An oligonucleotide comprising or consisting of a sequence having homology with a nucleotide sequence having a length of 10 - 50 bases comprising base positions 136 - 145 of the base sequence of SEQ ID NO: 3, and in which the base that is homologous to base number 145 is cytosine, and the cytosine is fluorescence-labeled.