Oligonucleotide Probes for Distinct Tm Peaks in Gene Polymorphism Detection
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Solution Overview
Problem
Current methods for detecting gene polymorphisms, particularly in immune-related genes like FCGR3A, FCGR2A, IL-10, TNF α, and TNF β, face challenges in distinguishing between homozygotes and heterozygotes due to overlapping signal peaks in melting curves, leading to impaired detection sensitivity and difficulty in determining specific polymorphisms.
Innovation Solution
Development of specific oligonucleotide probes that target distinct regions of these genes, allowing for clear differentiation of polymorphisms through Tm analysis by ensuring signal peaks are sufficiently distant, even in heterozygotes, enabling reliable detection of single-base differences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If conventional Tm analysis is used to detect gene polymorphisms, then the detection process is simplified and automation is enabled, but the ability to distinguish between homozygotes and heterozygotes is impaired due to overlapping signal peaks
Solution Approach 1:
The invention divides the detection approach by using multiple probes with different binding affinities (different Tm values) to segment the detection of different polymorphism types. The first probe detects homozygotes while the second probe detects heterozygotes, allowing clear distinction between the two states without peak overlap by using probes designed to bind with different thermal stabilities to the target sequence
2Ease of manufacture
If standard oligonucleotide probes are used for Tm analysis, then the detection method is straightforward, but single-base differences cannot be reliably distinguished due to peak overlap in melting curves
Solution Approach 1:
The invention applies local quality by designing probes with specific modifications at particular positions (such as introducing mismatches at specific locations or using locked nucleic acids at key positions) to enhance the Tm difference between matching and mismatching hybrids. This localized modification ensures that single-base differences produce distinct Tm shifts, enabling reliable detection while maintaining the overall simplicity of the Tm analysis approach
3Device complexity
If a single probe is used for polymorphism detection, then the detection process is simple, but multiple polymorphism types cannot be distinguished
Solution Approach 1:
The invention achieves universality by designing a multi-probe system where each probe is optimized for detecting specific polymorphism types (homozygotes vs. heterozygotes). The first probe with higher binding affinity detects homozygous sequences, while the second probe with lower binding affinity detects heterozygous sequences. This multi-functional probe set enables a single detection system to identify multiple polymorphism types across different genes (FCGR3A, FCGR2A, IL-10, TNF-α, TNF-β) without requiring separate assays for each type
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 enable accurate and reliable detection of immune-related gene polymorphisms, including heterozygotes, by ensuring distinct signal peaks, thereby improving the reliability of polymorphism identification and informing medical treatment decisions for antibody drugs.
Implementation Method 1
using a probe that is complementary to a region containing the SNP to be detected, a hybrid (double-strand nucleic acid) between a sample nucleic acid and the probe is formed
Implementation Method 2
The hybridization product then is subjected to heat treatment, and the dissociation (melting) of the hybrid into a single-strand nucleic acid in response to a temperature increase is detected
Data Source
AI summary
Polymorphism detection probes that can distinguish polymorphisms that have only one different base are provided. At least one oligonucleotide selected from the group consisting of the oligonucleotides of SEQ ID NOS. 4, 23, 30, 47, 57 and 64 is used as a probe in a Tm analysis. A Tm analysis using such probes allows easy detection of specific polymorphisms of the FCGR3A gene, the FCGR2A gene, the IL-10 gene, the TNF α gene and the TNF β gene that have an effect on the pharmaceutical effects of antibody drugs or the like. Moreover, such probes allow detection of two or more types of polymorphisms in a single reaction system by introducing two or more types of the probes concomitantly.