Fluorescence Oligonucleotide Probe for ABL Gene Mutation Detection
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for detecting point mutations in the ab1 gene, such as those used in leukemia therapy, face challenges including low sensitivity, labor-intensive processes, and difficulty in distinguishing between mutant and normal sequences, especially in the presence of both types in a sample, which complicates the detection of resistance to antileukemic agents like imatinib.
Innovation Solution
Designing specific fluorescence-labeled oligonucleotide probes that target particular regions of the ab1 gene, including T1076G, T757C, A764T, and G895C/T mutations, for use in melting curve analysis to enhance detection sensitivity and specificity, allowing for the differentiation of mutant and normal sequences even when they coexist.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional detection methods (sequencing, PCR-RFLP, DHPLC) are used to detect point mutations in the ab1 gene, then detection capability is achieved, but the process becomes labor-intensive and time-consuming with difficulty in automation
Solution Approach 1:
The patent replaces manual mechanical operations (cloning, gel electrophoresis, sequencing preparation) with an automated fluorescence-based detection system using real-time PCR and melting curve analysis. The fluorescence signal automatically tracks DNA hybridization and melting, eliminating the need for manual sample handling and visual analysis of gels or sequencing traces.
Solution Approach 2:
The patent utilizes changes in fluorescence parameters (intensity and wavelength) as temperature changes during melting curve analysis. By monitoring fluorescence emission at different temperatures, the system automatically detects mutation-specific melting temperatures, converting a manual visual inspection process into an automated parameter-based detection system.
2Measurement precision
If conventional detection methods are used, then mutation detection is possible, but the process takes several days to complete
Solution Approach 1:
The patent performs preliminary amplification of the target DNA region using PCR before detection, and uses melting curve analysis to pre-identify potential mutations. This preliminary fluorescence-based screening eliminates the need for time-consuming post-PCR steps such as gel electrophoresis, cloning, and sequencing, reducing total detection time from several days to a single day.
Solution Approach 2:
The patent extracts and eliminates the time-consuming intermediate steps from the conventional detection workflow. By removing cloning, gel electrophoresis, and sequencing preparation steps, and replacing them with direct fluorescence-based melting curve analysis, the patent reduces detection time while maintaining accuracy.
3Extent of automation
If Tm analysis is used for polymorphism detection, then automation becomes possible, but sensitivity decreases making it difficult to detect mutant sequences when both mutant and normal sequences coexist
Solution Approach 1:
The patent designs probes with specific local properties: they are labeled with fluorescent dyes at particular positions (5' or 3' end) and have specific lengths (10-50 nucleotides) to optimize hybridization to mutant sequences. The probes are designed to bind specifically to the mutation site region, creating local conditions that enhance mutant detection sensitivity even in the presence of normal sequences.
Solution Approach 2:
The patent utilizes the dynamic melting process of DNA-probe hybrids, where the fluorescence signal changes continuously as temperature increases and hybrids dissociate. By analyzing the dynamic fluorescence response during the melting transition, the system can distinguish mutant from normal sequences based on their different melting temperatures, improving sensitivity in mixed samples.
4Extent of automation
If Tm analysis with conventional probes is used, then detection is possible, but discrimination between mutant types becomes difficult requiring separate sequencing
Solution Approach 1:
The patent segments the detection process into multiple temperature ranges, analyzing fluorescence signals at different temperature intervals. By dividing the melting curve into distinct temperature zones, the system can identify different mutation types based on their characteristic melting temperatures, preserving mutation type information without requiring separate sequencing reactions.
Solution Approach 2:
The patent adds the temperature dimension to the fluorescence detection, creating a two-dimensional analysis (fluorescence intensity vs. temperature). This dimensional expansion allows the system to distinguish between different mutation types based on their unique melting temperature profiles, providing comprehensive mutation type discrimination within a single automated assay.
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 proposed solution significantly improves the sensitivity and specificity of detecting ab1 gene mutations, enabling effective monitoring of resistance to antileukemic agents and guiding therapeutic strategies by clearly separating the signals for mutant and normal sequences in melting curve analysis.
Implementation Method 1
a target single-stranded DNA in the detection sample is allowed to form a hybrid (double-stranded DNA) with the probe
Implementation Method 2
dissociation (melting) of the hybrid due to increased temperature is detected by measurement of a signal such as the absorbance
Data Source
AI summary
A probe for detecting a polymorphism in ab1 gene, comprising at least one fluorescence-labeled oligonucleotide.