Allele Specific Primer Design for Accurate SNP Discrimination
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Solution Overview
Problem
Existing SNP discrimination methods using allele specific primers face challenges with false positives due to inefficient primer extension reactions, particularly when the 3′ end base of the primer is noncomplementary to the target SNP base, and the efficiency of reactions varies with the second and third base types from the 3′ end.
Innovation Solution
A method involving an allele specific primer with a 3′ end base complementary to one of four possible bases (A, T, G, or C) and second and third bases from the 3′ end as 5′-AT-3′, 5′-GT-3′, 5′-CA-3′, or 5′-CT-3′, which binds efficiently when the target SNP base is complementary, allowing for accurate discrimination by examining the efficiency of the DNA elongation reaction.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If the 3′ end base of the allele specific primer is made complementary to the target SNP base, then the primer extension reaction efficiency is improved, but false positives occur when the base is noncomplementary
Solution Approach 1:
The patent applies local quality by making only the 3′ end base of the primer complementary to the target SNP base, while the rest of the primer sequence remains completely complementary to the target DNA sequence. This localized complementarity at the critical 3′ end position enables selective extension only when the base matches, improving both efficiency and reliability without requiring strict control of reaction conditions.
2Reliability
If reaction conditions are strictly defined to prevent false positives, then reliability is improved, but device complexity and operation difficulty increase
Solution Approach 1:
The patent implements self-service by designing the allele specific primer with a 3′ end base that inherently provides self-regulation of the extension reaction. The primer structure itself ensures that extension occurs only when the 3′ end base is complementary to the target SNP base, eliminating the need for complex external control mechanisms or strict definition of reaction conditions such as temperature, time, and dNTP concentration.
3Reliability
If the second and third bases from the 3′ end are made noncomplementary to reduce false positives, then reliability is improved, but primer extension efficiency decreases
Solution Approach 1:
The patent applies local quality by restricting the complementarity requirement only to the 3′ end base position, while allowing the second and third bases from the 3′ end to be noncomplementary. This localized approach maintains high extension efficiency when the 3′ end base matches the target SNP base, while the noncomplementary bases at other positions prevent false positives when the 3′ end base does not match.
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 significantly reduces the possibility of false positives and enables definite SNP discrimination by differentiating between complementary and noncomplementary target bases through high or low efficiency of the primer extension reaction, respectively.
Implementation Method 1
a base sequence consisting of the second and the third bases from the 3′ end of the allele specific primer is 5′-AT-3′, 5′-GT-3′, 5′-CA-3′ or 5′-CT-3′, and a base sequence between the fourth base from the 3′ end and the 5′ end base of the allele specific primer complementarily binds to a base sequence between the base on the DNA corresponding to the fourth base from the 3′ end of the allele specific primer and the base on the DNA corresponding to the 5′ end base of the allele specific primer
Implementation Method 2
a DNA elongation step comprising binding to the DNA an allele specific primer having at its 3′ end a base that is complementary to a base predicted as the target base among 4 bases of A, T, G and C to cause a DNA elongation reaction
Data Source
AI summary
An object of the present invention is to provide an allele specific primer which is accompanied by less possibility of the false positive and enables definite discrimination when a base immediately adjacent to on the 3′ side of a target SNP base is C, while a base adjacent with one base spaced apart is A.According to the present invention, the 3′ end base is designed to be the base corresponding to SNP; the second and the third bases from the 3′ end to be 5′-AT-3′, 5′-GT-3′, 5′-CA-3′ or 5′-CT-3′; and the base sequence of from the fourth from the 3′ end to the 5′ end base to be completely complementary to the sequence of from a base three bases away from the target SNP base on the 3′ side to a desired base.