Loopable Primers for Selective Nucleic Acid Amplification
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current nucleic acid amplification methods face challenges such as inadvertent primer dimerization and spurious internal priming, especially when using random or degenerate primers, which can lead to inefficient amplification and the dominance of short fragment artifacts.
Innovation Solution
The use of loopable primers with a universal region and optionally a noncomplementary region within the loop, allowing for the selective amplification of target nucleic acid sequences by reducing nonspecific hybridization and primer dimer formation, and enabling the amplification of larger sections over smaller sections.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If random or degenerate primers are used for amplification, then amplification coverage is improved, but primer dimerization and spurious internal priming increase
Solution Approach 1:
The primer is divided into distinct functional segments: a target-binding region for specific annealing and a loop-forming region with non-complementary sequences. This segmentation allows the primer to perform multiple functions while preventing self-hybridization, as the non-complementary loop region cannot form stable dimers with other primers.
Solution Approach 2:
The loop-forming region acts as an intermediary structure that prevents direct interaction between target-binding regions of different primers. By forcing primers to form intra-molecular loops rather than inter-molecular dimers, this intermediary structure eliminates spurious priming while preserving amplification coverage.
2Manufacturing precision
If conventional PCR methods are used, then specific sequence amplification is achieved, but amplification of larger genome sections is limited
Solution Approach 1:
The primer structure is designed to be dynamic rather than static. The loop-forming region can adopt different conformations during the amplification process, allowing the primer to bind to target sequences and facilitate extension of longer fragments. This dynamic structure enables the primer to adapt to various target lengths while maintaining specificity.
Solution Approach 2:
The invention changes the structural parameters of the primer by introducing loop-forming regions with specific nucleotide compositions and lengths. These parameter changes allow the primer to stabilize during extension reactions, enabling efficient amplification of larger genome sections while maintaining the precision of conventional PCR for specific sequences.
3Reliability
If loopable primers with noncomplementary regions are used, then nonsspecific hybridization is reduced, but primer design complexity increases
Solution Approach 1:
Different regions of the primer are assigned different local qualities: the target-binding region has high complementarity for specific annealing, while the loop-forming region has non-complementary sequences to prevent self-hybridization. This local differentiation of quality allows the primer to achieve high reliability in specific hybridization while managing design complexity through region-specific optimization.
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 enhances the efficiency of nucleic acid amplification by reducing nonspecific binding and primer dimerization, allowing for the selective amplification of larger target sequences while minimizing the amplification of shorter, undesired sequences, thereby improving the accuracy and yield of the amplification process.
Implementation Method 1
the loopable primer includes a random region, a first loop forming region, a universal region, optionally a noncomplementary region, and a second loop forming region. The first and second loop forming regions include nucleic acid sequences that allow the regions to hybridize to one another.
Implementation Method 2
a sequence associated with a target sequence that has been amplified by the loopable primer (a double-extended loopable primer) forms a self-hybridized structure involving the noncomplementary region and/or the universal region.
Data Source
AI summary
The present disclosure relates to the amplification of target nucleic acid sequences. This can be accomplished via the use of various primers. The use of these primers, as described herein, results in nucleic acid structures that can reduce the amplification of nonspecific hybridization events (such as primer dimerization) while allowing the amplification of the target nucleic acid sequences.