Primer 3' Non-Complementary Moiety for Nucleic Acid Amplification
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Solution Overview
Problem
Nucleic acid amplification reactions often generate non-specific amplification products such as primer dimer by-products, which reduce the efficiency and specificity of the reaction.
Innovation Solution
The use of primers with a molecular moiety at the 3' end that is non-complementary to the target nucleic acid, allowing extension only after removal of the moiety, and a primer set where the forward and reverse primers are non-complementary to each other, minimizing primer dimer formation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional primers are used in nucleic acid amplification, then the amplification reaction can proceed, but non-specific amplification products such as primer dimer by-products are generated that reduce efficiency and specificity
Solution Approach 1:
The patent extracts the problematic 3' end of the primer that causes non-specific binding and primer dimer formation. By removing this region or replacing it with a non-complementary molecular moiety, the harmful primer dimer by-products are eliminated while preserving the amplification function through the remaining complementary sequence
Solution Approach 2:
The primer is divided into two functional segments: a 5' portion containing the nucleotide sequence complementary to the target nucleic acid that enables specific binding, and a 3' portion containing a molecular moiety that is non-complementary and prevents unwanted extension. This segmentation allows the primer to maintain specificity while avoiding primer dimer formation
2Productivity
If primers with extended 3' ends are used to increase amplification efficiency, then more binding sites are available, but the likelihood of non-specific binding and primer dimer formation increases
Solution Approach 1:
Different regions of the primer are assigned different functional qualities: the 5' region possesses complementary nucleotide sequence for specific target binding, while the 3' region contains a non-complementary molecular moiety that prevents non-specific extension. This local differentiation allows the primer to bind specifically without generating harmful by-products
Solution Approach 2:
The molecular moiety at the 3' end acts as an intermediary element that mediates between the need for primer stability and the need to prevent non-specific binding. It provides a structural feature that blocks unwanted interactions while allowing the complementary 5' region to maintain specific binding to the 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
This approach significantly reduces the concentration of primer dimer by-products to less than 10% of the amplified target nucleic acids, enhancing the specificity and efficiency of the amplification process.
Implementation Method 1
a molecular moiety at the 3' end that is non-complementary with respect to one or more corresponding nucleotides of the target nucleic acid molecule... adapted to be extended in a primer extension reaction to form a complement nucleic acid strand of the target nucleic acid molecule only upon removal of the molecular moiety
Implementation Method 2
the 3' end may be cleavable by an enzyme with 3' to 5' exonuclease activity
Data Source
AI summary
An embodiment relates to system comprising: (i) a reaction mixture comprises: a) a target nucleic acid molecule; b) a forward primer complementary to a strand of the target nucleic acid molecule, the forward primer comprises a first molecular moiety at a 3′ end, wherein the first molecular moiety is non-complementary to the strand of the target nucleic acid molecule; c) a reverse primer complementary to a complementary sequence of the strand of the target nucleic acid molecule, the reverse primer comprises a second molecular moiety at a 3′ end, wherein the second molecular moiety is non-complementary to the complementary sequence of the strand of the target nucleic acid molecule; d) a polymerase with 3′-5′ exonuclease activity; and (ii a device suitable of detecting an amplification products.