SNV Detection Primer With Non-Complementary 3' End Nucleotides
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Solution Overview
Problem
Conventional methods for detecting rare single-nucleotide variants (SNVs) suffer from low specificity and sensitivity, requiring extensive time and resources, and lack the capability to accurately analyze trace amounts.
Innovation Solution
A novel primer composition for PCR that includes a first primer complementary to the DNA fragment with a single nucleotide variant and additional non-complementary nucleotides at the 3' end, along with a second phosphorylated primer for wild-type DNA, allowing for specific and sensitive detection of SNVs using DNA polymerase without 3'->5' exonuclease activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional methods (MALDI mass spectrometry, DNA sequencing, microarrays) are used for detecting single-nucleotide variants, then detection capability is achieved, but specificity for trace amount SNV is low and sensitivity is poor
Solution Approach 1:
The primer is designed with non-complementary nucleotides at the 3' end specifically positioned to create mismatch with wild-type DNA while maintaining complementarity with mutant DNA. This localized modification at the primer's 3' end enables selective amplification of trace mutant alleles without amplifying abundant wild-type DNA, thereby improving detection specificity for trace SNV
Solution Approach 2:
The invention changes the nucleotide composition parameter of the primer by introducing 2-6 non-complementary nucleotides at the 3' end. This parameter modification creates differential binding affinity between the primer and wild-type versus mutant DNA templates, enabling the PCR reaction to selectively amplify trace mutant DNA even in the presence of abundant wild-type DNA
2Measurement precision
If conventional methods are used for detecting single-nucleotide variants, then detection is performed, but detection efficiency is low due to poor sensitivity
Solution Approach 1:
The primer design introduces non-complementary nucleotides specifically at the 3' end region, creating a localized mismatch that prevents extension by DNA polymerase when binding to wild-type DNA. This localized modification enables highly sensitive detection of trace mutant alleles by selectively allowing amplification only of mutant templates, dramatically improving detection sensitivity and efficiency
3Measurement precision
If conventional methods are used for detecting single-nucleotide variants, then analysis is performed, but it takes a lot of time and high cost
Solution Approach 1:
The invention modifies the primer's nucleotide sequence parameter by adding non-complementary nucleotides at the 3' end, which enables the PCR reaction to selectively and rapidly amplify mutant DNA. This parameter change allows for fast, accurate detection of trace SNV within a single PCR cycle, significantly reducing both detection time and cost compared to conventional methods that require multiple steps or specialized equipment
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 primer composition enables highly specific and sensitive detection of rare SNVs, reducing the complexity and cost of the detection process while effectively amplifying only the target SNV, even in trace amounts.
Implementation Method 1
a first primer that includes a nucleotide sequence complementary to the to-be-detected DNA fragment including a single nucleotide variant
Implementation Method 2
performing a polymerase chain reaction (PCR) using the primer composition for detecting a single-nucleotide variant
Data Source
AI summary
The present invention relates to a primer set for detecting a trace amount of a rare single-nucleotide variant and a method for specifically and sensitively detecting a trace amount of a rare single-nucleotide variant by using same. Particularly, the present invention relates to: a primer composition for detecting a single-nucleotide variant (SNV), the primer composition comprising a first primer that comprises a nucleotide sequence complementary to a to-be-detected DNA fragment comprising an SNV to be detected, and at the 3′ end of the primer, 2-6 nucleotide sequences that are not complementary to the to-be-detected DNA fragment; and a method for detecting a trace amount of a rare single-nucleotide variant, the method comprising performing a polymerase chain reaction (PCR) using the composition.