Mutation Detection via Primer Tm Adjustment
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Solution Overview
Problem
Current methods for detecting mutations, such as direct sequencing, ASP-PCR, and Tm analysis, face challenges with low sensitivity, high specificity issues, and the need for multiple reaction systems, making it difficult to accurately detect mutations in samples with low mutant gene content, particularly in clinical settings where both normal and mutant genes are present.
Innovation Solution
A method for amplifying target nucleic acid sequences that preferentially amplifies mutant-type sequences over normal-type sequences in a single reaction system, using primers with adjusted Tm values and lengths to enhance specificity and sensitivity, allowing for reliable mutation detection using Tm analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If direct sequencing method is used, then measurement precision is improved, but productivity deteriorates due to considerable time and effort required
Solution Approach 1:
The invention extracts and analyzes only the specific base site region containing the mutation rather than sequencing the entire gene sequence. This is achieved by designing primers that specifically amplify only the region containing the objective base site, thereby reducing the time and effort required while maintaining detection accuracy for the mutation of interest
Solution Approach 2:
The invention uses fluorescently labeled probes that emit different signals based on hybridization status with mutant or normal sequences. The probe fluorescence intensity changes indicate the presence or absence of mutation, enabling rapid detection without time-consuming sequencing operations
2Productivity
If ASP-PCR method is used, then productivity is improved, but reliability deteriorates due to low specificity and false positives
Solution Approach 1:
The invention introduces a probe as an intermediary element that hybridizes to the amplification product to confirm mutation presence. The probe acts as a mediator between the PCR amplification and final detection, providing an additional layer of verification that reduces false positives while maintaining operational efficiency
Solution Approach 2:
The invention optimizes the Tm values of primers and probes to ensure specific binding only to mutant sequences. By carefully controlling the annealing temperature and Tm parameters, the method achieves high specificity that prevents false positives while maintaining the speed advantage of PCR-based methods
3Reliability
If Tm analysis method is used, then reliability is improved, but productivity deteriorates due to insufficient sensitivity
Solution Approach 1:
The invention performs PCR amplification of the target region before conducting Tm analysis. This preliminary amplification step concentrates the mutant sequences from the original sample, thereby enhancing the sensitivity of subsequent Tm analysis while maintaining the high specificity that Tm analysis provides
Solution Approach 2:
The invention combines PCR amplification within Tm analysis in a nested structure. The PCR reaction is performed first to amplify the target sequences, and then the amplified products are subjected to Tm analysis with probes. This nested approach allows the sensitive detection capability of PCR to enhance the specific detection capability of Tm analysis
4Reliability
If multiple reaction systems are used for ASP-PCR, then reliability is improved, but device complexity increases
Solution Approach 1:
The invention combines multiple detection capabilities into a single reaction system by using both forward and reverse primers with different Tm values in the same PCR reaction. This allows simultaneous detection of different mutations or confirmation of mutant sequences without requiring separate reaction systems, thereby reducing operational complexity while maintaining detection reliability
Solution Approach 2:
The invention designs a universal probe that can hybridize to various mutant sequences with different Tm characteristics. This universal probe system can detect multiple types of mutations using the same basic reaction setup, eliminating the need for multiple specialized reaction systems and reducing overall system complexity
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 approach enables high sensitivity and reliability in detecting mutations even when mutant genes are present in low proportions, facilitating accurate diagnosis and treatment in clinical settings by utilizing a single reaction system and reducing the risk of false positives.
Implementation Method 1
a hybrid (double-stranded DNA) between the thus obtained amplification product and a probe that is complementary to the mutant sequence is formed
Implementation Method 2
dissociation (melting) of the hybrid accompanying the temperature rise is detected
Data Source
AI summary
The present invention provides a method for detecting a mutation capable of detecting a mutation with high sensitivity and high reliability in one reaction system. Using primers (Xmt) and (Xwt), a target nucleic acid sequence whose objective base to be detected is a mutant-type is amplified with amplification efficiency higher than a target nucleic acid sequence whose objective base to be detected is a normal-type. The (Xmt) is a primer that is complementary to a region including a mutant-type base in the template nucleic acid and has a base complementary to a mutant-type base at a 3′ region, and the (Xwt) is a primer that is complementary to a region including a normal-type base in the template nucleic acid and has a base complementary to a normal-type base at a 3′ region. It is preferable that amplification efficiency by the (Xmt) with reference to a mutant-type template nucleic acid is higher than that by the (Xwt) with reference to a normal-type template nucleic acid. Then, a signal value that shows a molten state of a hybridization product between the thus obtained amplification product and the probe is measured, and the presence or absence of the mutation of the objective base site is determined from a change in the signal value accompanying a change in the temperature.