Oligonucleotide Probe for K-ras Polymorphism Detection via Melting Temperature
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Solution Overview
Problem
Current methods for detecting polymorphisms in the K-ras gene, such as PCR-RFLP and Tm analysis, face challenges including cumbersome procedures, risk of sample dispersion, and impractical analysis of multiple samples, especially when both wild-type and mutant-type polymorphisms are present together.
Innovation Solution
A probe comprising an oligonucleotide sequence (e.g., SEQ ID NO: 10 or 31) is used for detecting polymorphisms in the K-ras gene, allowing for reliable identification through Tm analysis, even in samples with both wild-type and mutant-type polymorphisms, by hybridizing with the amplified gene product and measuring the melting temperature.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If PCR-RFLP method is used for detecting polymorphisms, then polymorphism detection can be performed, but the procedure becomes cumbersome and sample handling becomes complex
Solution Approach 1:
The invention extracts only the essential detection function by using a specifically designed oligonucleotide probe that directly hybridizes to the polymorphic site. This eliminates the need for restriction enzyme digestion and Southern hybridization steps, retaining only the critical hybridization-based detection while removing cumbersome intermediate procedures.
Solution Approach 2:
The invention introduces a specifically designed oligonucleotide probe as an intermediary element that mediates between the target DNA and the detection system. This probe contains a mismatch at the polymorphic position, enabling direct detection through hybridization stability differences without requiring complex enzymatic reactions or gel electrophoresis.
2Reliability
If Tm analysis is used for polymorphism detection, then detection can be performed, but analysis of multiple samples becomes impractical
Solution Approach 1:
The invention changes the detection parameter from measuring melting temperature (Tm) to measuring hybridization signal intensity or presence/absence. This parameter change allows for high-throughput detection using standard hybridization assays, real-time PCR, or microarray technologies, making it practical to analyze multiple samples efficiently while maintaining detection reliability.
3Reliability
If conventional detection methods are used, then polymorphism detection is possible, but the risk of sample dispersion and contamination increases
Solution Approach 1:
The invention merges the amplification and detection steps into a single integrated assay. The oligonucleotide probe is designed to work directly with PCR amplification products or even in real-time during amplification, eliminating the need to transfer samples between tubes and reducing opportunities for dispersion and contamination while maintaining detection accuracy.
4Adaptability or versatility
If multiple probes are used to detect different polymorphisms, then detection coverage improves, but device complexity increases
Solution Approach 1:
The invention designs a universal oligonucleotide probe structure that can detect any polymorphism by simply changing the sequence at the polymorphic position. This single probe design paradigm can be applied to detect any single-nucleotide polymorphism, making the system highly versatile without requiring complex multi-probe systems. The probe structure itself is universal, only the specific sequence needs to be customized for each target.
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 enables easy and reliable detection of polymorphisms in the K-ras gene, improving diagnostic accuracy and treatment selection for diseases associated with these mutations, particularly in samples with mixed wild-type and mutant-type sequences.
Implementation Method 1
using a probe complementary to a region including a detection target polymorphism, a hybrid (double-stranded nucleic acid) of a nucleic acid to be examined (hereinafter simply referred to as a 'test nucleic acid') with the probe is formed
Implementation Method 2
the thus-obtained hybrid is heat-treated, and dissociation (melting) of the hybrid into single-stranded nucleic acids accompanying the temperature rise is detected
Data Source
Figure 1A~1E
Figure 2A~2E
Figure 3A~3C
AI summary
The present invention provides a polymorphism detection probe that can identify a different polymorphism in a K-ras gene easily with high reliability and use of the polymorphism detection probe. The polymorphism detection probe is a probe for detecting a polymorphism in a K-ras gene that is a disease-related gene, including at least one of oligonucleotides (P1), (P2), (P3), (P1'), (P2'), and (P3'): (P1) a 11- to 50-mer oligonucleotide that is composed of a base sequence complementary to a base sequence including the 220th to 230th bases in SEQ ID NO: 1 and has a base complementary to the 230th base in its 5' end region; (P1') an oligonucleotide composed of a base sequence complementary to the oligonucleotide (P1); (P2) a 15- to 50-mer oligonucleotide that is composed of a base sequence complementary to a base sequence including the 220th to 234th bases in SEQ ID NO: 1 and has a base complementary to the 234th base in its 5' end region; (P2') an oligonucleotide composed of a base sequence complementary to the oligonucleotide (P2); (P3) a 17- to 50-mer oligonucleotide that is composed of a base sequence complementary to a base sequence including the 220th to 236th bases in SEQ ID NO: 1 and has a base complementary to the 236th base in its 5' end region; and (P3') an oligonucleotide composed of a base sequence complementary to the oligonucleotide (P3).