Multiplex PCR Primer Design for 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 artifacts such as primer-dimers and superamplicons, especially when amplifying thousands of targets in a single reaction, which can lead to inefficient downstream applications like next-generation sequencing.
Innovation Solution
The use of target-specific primers with cleavable groups and adapter-ligation techniques allows for the selective amplification of multiple targets in a single reaction, reducing the formation of artifacts through controlled primer interactions and efficient amplification protocols.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiple target sequences are amplified in a single multiplex PCR reaction, then the productivity and efficiency of downstream applications are improved, but the formation of amplification artifacts such as primer-dimers and superamplicons increases
Solution Approach 1:
The patent divides the amplification process into two separate reactions: a first PCR reaction that generates initial amplicons, and a second PCR reaction that amplifies those amplicons. This segmentation prevents primer-dimer and superamplicon formation in the second reaction since primers specific to the first reaction are used, thereby resolving the contradiction between high productivity and artifact formation.
2Quantity of substance
If conventional multiplex PCR is used to amplify multiple targets, then the quantity of amplicons produced is increased, but the specificity and yield for downstream applications deteriorate due to artifact formation
Solution Approach 1:
The two-stage PCR approach segments the amplification process, allowing the first reaction to generate sufficient quantity of amplicons while the second reaction, using target-specific primers, ensures high specificity by avoiding artifact formation. This resolves the contradiction between quantity and precision.
Solution Approach 2:
The first PCR reaction performs preliminary amplification to generate adequate amounts of amplicons before the second reaction. This preliminary action ensures sufficient quantity is available for downstream applications while the subsequent specific amplification maintains high precision.
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 enables the simultaneous amplification of numerous target sequences with reduced artifact formation, improving the yield and specificity of amplicons, and facilitating accurate copy number variation analysis and sequencing applications.
Implementation Method 1
amplifying a plurality of target sequences within a sample by producing a plurality of different amplified target sequences in a single amplification reaction mixture by contacting the plurality of different target sequences with a plurality of targets-specific primers and a polymerase under amplification conditions
Implementation Method 2
cleaving the cleavable group from at least one amplified target sequence
Data Source
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.