Fluorescence-Based Competitive Hybridization for High-Throughput Mutation Detection
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Solution Overview
Problem
Current methods for detecting DNA mutations, such as DNA sequencing and gel electrophoresis, are inefficient for high-throughput mutation detection due to their slow speed and high cost, and existing hybridization-based methods are not easily multiplexed or sensitive enough to detect single base mismatches.
Innovation Solution
A fluorescence-based competitive hybridization method that uses labeled polynucleotide sequences to determine differences by preferential hybridization to a target sequence, allowing for multiplexing and automation without the need for nucleotide sequencing or gel electrophoresis, utilizing a mutation detection index (MDI) ratio to indicate sequence differences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If DNA sequencing is used for mutation detection, then measurement precision is improved, but productivity deteriorates due to low speed and high cost
Solution Approach 1:
The patent replaces traditional mechanical sequencing methods with a fluorescence-based optical detection system. Competitive hybridization reactions are monitored using fluorescently labeled probes and optical readers, eliminating the need for physical sequencing operations while maintaining mutation detection accuracy and enabling high-throughput parallel processing
Solution Approach 2:
The patent combines multiple functions into a single hybridization-based assay: mutation detection, genotyping, and sequence verification are all achieved through competitive hybridization reactions. This consolidation eliminates separate sequencing steps while maintaining measurement precision and improving productivity through multiplexing
2Ease of operation
If gel electrophoresis is used for mutation detection, then ease of operation is improved, but productivity deteriorates due to slow processing speed
Solution Approach 1:
The patent replaces gel electrophoresis with a fluorescence-based detection system that reads hybridization reactions in solution or on solid supports. This substitution eliminates time-consuming gel running and analysis steps while maintaining ease of operation through standardized fluorescent assay protocols that can be automated
Solution Approach 2:
The patent extracts the essential detection function from gel electrophoresis by using fluorescently labeled probes that directly report hybridization status through fluorescence intensity or ratio measurements. This extraction removes the slow electrophoresis step while preserving the ability to detect sequence differences
3Productivity
If hybridization-based methods are used for mutation detection, then productivity is improved, but measurement precision deteriorates due to inability to detect single base mismatches
Solution Approach 1:
The patent applies local quality by designing fluorescent probes with specific properties optimized for mismatch detection. Different probe types (e.g., perfectly matched vs. mismatched probes, different fluorescent labels, different probe lengths) are used in the competitive hybridization to enhance sensitivity to single base differences while maintaining high throughput capability
Solution Approach 2:
The patent uses fluorescent color changes or intensity ratios to detect single base mismatches. Different fluorescent labels (e.g., FITC, TRITC, Cy5) or fluorescence intensity ratios between test and driver sequences provide sensitive discrimination of sequence differences while enabling multiplexed high-throughput detection
4Ease of manufacture
If traditional hybridization methods are used, then ease of manufacture is improved, but adaptability deteriorates due to difficulty in multiplexing
Solution Approach 1:
The patent creates a universal fluorescent hybridization platform that can detect multiple different mutations, SNPs, or sequence variations using the same basic assay format. By using fluorescently labeled probes with different spectral properties or targeting different sequences in the same reaction, the system achieves multiplexing capability while maintaining ease of manufacture through standardized protocols
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 method enables rapid and efficient detection of DNA sequence differences, including single nucleotide polymorphisms, with enhanced sensitivity and multiplexing capabilities, overcoming the limitations of existing technologies by using fluorescence signals to differentiate between test and driver sequences.
Implementation Method 1
competitive hybridization between polynucleotide sequences
Implementation Method 2
fluorescence-based technology in the assessment of the results of competitive hybridization
Data Source
AI summary
The present invention relates generally is a method for determining the likelihood that a test polynucleotide sequence differs from a driver polynucleotide sequence. More particularly, the present method uses fluorescence-based technology in the assessment of the results of competitive hybridization between polynucleotide sequences. The present method does not require nucleotide sequencing or gel electrophoresis and is capable of being multiplexed and automated. The methods of the present invention will find broad application in the analysis of polynucleotides, inter alia in genetic analysis, specific locus testing, genotyping, mutation detection, the discovery and detection of single nucleotide polymorphisms (SNPs) and mapping.