Degradable Primers for Multiplex PCR Artifact Reduction
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Solution Overview
Problem
Current methods for multiplex PCR face challenges in selectively amplifying multiple target nucleic acid molecules while minimizing the formation of amplification artifacts such as primer-dimers and superamplicons, especially when amplifying thousands of targets in a single reaction, which complicates downstream applications like next-generation sequencing.
Innovation Solution
The use of degradable amplification primers and adapter-ligation techniques, where primers with cleavable groups are used to reduce primer-dimer formation and allow for efficient amplification of multiple targets in a single reaction, followed by adapter ligation and reamplification to enhance specificity and reduce non-specific products.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiple target sequences are amplified within a single amplification reaction, then productivity increases, but amplification artifacts such as primer-dimers and superamplicons increase
Solution Approach 1:
The patent modifies the chemical structure of primers by incorporating cleavable groups (such as ribose residues in DNA primers or deoxyribose residues in RNA primers) that can be selectively removed after amplification. This parameter change in primer composition allows multiple targets to be amplified simultaneously while providing a mechanism to eliminate artifact formation through selective cleavage of non-specific products.
Solution Approach 2:
The patent extracts or removes the harmful cleavable groups from the amplified products through selective cleavage. By using enzymes or chemical treatments that specifically remove cleavable groups from primer-dimers and superamplicons but not from specific amplicons, the harmful artifacts are separated and eliminated from the reaction mixture, leaving only the desired amplification products.
2Object-generated harmful factors
If degradable amplification primers with cleavable groups are used, then amplification artifact formation is reduced, but primer design and reaction complexity increases
Solution Approach 1:
The patent segments the primer structure into distinct functional regions: a target-specific binding region and a cleavable group region. This segmentation allows the primer to perform multiple functions - specific target recognition and artifact elimination - while simplifying the overall design process by separating the complexity of artifact reduction from target selection.
Solution Approach 2:
The cleavable group acts as an intermediary element between the primer and the amplification artifact. It mediates the reduction of primer-dimer formation by providing a selective handle for artifact removal, while itself being part of the primer structure. This intermediary approach simplifies the solution by introducing a single modular element that addresses the complexity of artifact management.
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 primer-dimer formation and increases the yield of specific amplicons, enabling the simultaneous amplification of thousands of target sequences with improved specificity and reproducibility, suitable for downstream applications like copy number variation analysis and sequencing.
Implementation Method 1
amplifying a plurality of different target sequences flanking one or more SNPs in a sample, comprising: a) producing a plurality of different amplified target sequences flanking the one or more SNPs within a single amplification reaction mixture, by contacting the plurality of different target sequences flanking the one or more SNPs with a plurality of target-specific primers and a polymerase under amplification conditions
Implementation Method 2
b) cleaving the cleavable group from at least one amplified target sequence
Implementation Method 3
c) producing one or more adapter-ligated amplified target sequences, by ligating at least one adapter to at least one amplified target sequence
Data Source
Figure 1A~1C1
Figure 1D1~1E1
Figure 1E1i~1E2
AI summary
The present invention provides methods, compositions, kits, systems and apparatus that are useful for determining copy number variation of one or more nucleic acids present in a sample. In some aspects, the method includes various target-specific primers that allow for the selective amplification of one or more target nucleic acids in the sample. In yet another aspect, the invention relates to determining copy number variation with respect to gene or chromosome representation of a nucleic acid in the sample. In some aspects, the method for determining copy number variation of different target nucleic acids in a sample using the disclosed methods, kits, systems and apparatuses can be used in various downstream processes including diagnosis, predictive therapeutic regimes or other therapeutic purposes.