Polymorphism Detection Primer With Inhibitory Oligonucleotide
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Solution Overview
Problem
Current SNP detection methods, such as SSP-PCR and ASP-PCR, face challenges in sensitivity due to low flexibility in primer design, and non-amplification methods like strand displacement reactions suffer from insufficient sensitivity and accuracy, especially with small nucleic acid samples.
Innovation Solution
Incorporating an inhibitory oligonucleotide that hybridizes with the 5' side of the detection primer, preventing nonspecific nucleic acid extension reactions and enhancing the accuracy of polymorphism identification by stabilizing specific hybrids over nonspecific ones.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If SSP-PCR or ASP-PCR methods are used to detect genetic polymorphism, then signal amplification is achieved enabling detection in small nucleic acid samples, but sensitivity is insufficient due to low flexibility in primer design
Solution Approach 1:
The detection primer is divided into two functional regions: a 3' end region that specifically hybridizes to the polymorphic site for accurate detection, and a 5' end region that hybridizes to the inhibitory oligonucleotide for signal amplification. This segmentation allows the primer to simultaneously achieve specific binding and amplification capability.
Solution Approach 2:
An inhibitory oligonucleotide is introduced as an intermediary molecule that does not hybridize to the target nucleic acid but instead binds to the 5' end of the detection primer. This intermediary enables amplification by preventing nonspecific extension while allowing specific polymorphism detection at the 3' end.
2Adaptability or versatility
If non-amplification methods like strand displacement reactions are used, then primer design flexibility is improved, but sensitivity is insufficient for detecting polymorphisms in samples with very small nucleic acid amounts
Solution Approach 1:
The inhibitory oligonucleotide is designed to preliminarily bind to the 5' end of the detection primer before the polymorphism detection occurs. This preliminary binding prevents nonspecific extension reactions and creates a controlled environment that enhances sensitivity for low-abundance targets while maintaining design flexibility.
3Adaptability or versatility
If strand displacement reactions are used for polymorphism identification, then primer design flexibility is improved, but identification accuracy is insufficient due to easy hybridization with non-target sequences
Solution Approach 1:
Different regions of the detection primer are assigned different functional qualities: the 3' end region is optimized for specific hybridization to the polymorphic site to ensure accurate identification, while the 5' end region is designed to hybridize with the inhibitory oligonucleotide for amplification. This local differentiation of function resolves the accuracy problem while maintaining flexibility.
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 improves the sensitivity and accuracy of polymorphism detection, particularly in clinical samples with low nucleic acid concentrations, allowing for the detection of somatic mutations that were previously difficult to identify.
Implementation Method 1
Incorporating an inhibitory oligonucleotide that hybridizes with the 5' side of the detection primer
Implementation Method 2
performing a nucleic acid chain extension reaction with use of a nucleic acid in a test nucleic acid sample as a template, a type I detection primer, and a polymerase
Data Source
AI summary
The present invention is to provide a method for identifying a polymorphism. The method includes performing a nucleic acid chain extension reaction and identifying the polymorphism of the nucleic acid contained in the test nucleic acid sample. The extension reaction is conducted with use of a nucleic acid in a test nucleic acid sample as a template, a type I detection primer which hybridizes with a region including the polymorphic site of a nucleic acid whose polymorphic site nucleotide sequence consisting of a first nucleotide sequence, and a polymerase. The reaction is conducted with the presence of an inhibitory oligonucleotide, which hybridizes with the type I detection primer. The region of the type I detection primer to hybridize with the inhibitory oligonucleotide is located on the 5′ side of the polymorphism detection site of the type I detection primer to hybridize with the polymorphic site.