Multiplex PCR Primers with Cleavable Adapters 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 reactions and contamination in downstream applications.
Innovation Solution
The use of target-specific primers with cleavable groups and adapters that are ligated to amplified sequences, allowing for selective amplification and subsequent digestion to reduce non-specific products, along with strategies to minimize primer-primer interactions and optimize primer design to prevent artifact formation.
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 the amplification process is 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 distinct stages: a first amplification reaction that generates initial amplicons, and a second amplification reaction that selectively amplifies only the desired target sequences using adapter-ligated primers. This segmentation prevents artifacts generated in the first stage from being amplified in the second stage, thereby resolving the contradiction between high productivity and artifact formation.
Solution Approach 2:
The patent extracts and removes amplification artifacts from the reaction mixture through selective amplification strategies. By designing primers that specifically recognize adapter sequences ligated to genuine target amplicons, the method extracts only the desired products while leaving artifacts behind, which are then discarded in the waste stream.
2Quantity of substance
If primer concentration is increased to amplify more targets, then the coverage and completeness of target amplification is improved, but primer-primer interactions and artifact formation increase
Solution Approach 1:
The patent introduces adapter sequences as intermediary elements that mediate between the initial amplification products and the selective amplification step. These adapters serve as unique identifiers that allow high concentrations of primers to be used in the first reaction without causing excessive artifact formation, because the second reaction uses adapter-specific primers that only amplify genuine targets.
3Productivity
If secondary amplification is performed for downstream sequencing applications, then the yield of amplification product is improved, but amplification artifacts are further amplified and carried through the process
Solution Approach 1:
The patent converts the harmful effect of artifact formation into a beneficial selection mechanism. By designing the system so that only genuine target amplicons receive adapter ligations and subsequent primer bindings, artifacts are naturally excluded from further amplification. The artifacts that do form in the first reaction serve as negative controls that help identify successful target amplification events.
4Reliability
If primer design is optimized for specificity, then the formation of non-specific amplification products is reduced, but the number of targets that can be amplified in a single reaction is limited
Solution Approach 1:
The patent makes the adapter sequences universal and multi-functional, serving as common binding sites for all target-specific primers in the second amplification reaction. This allows highly specific target primers to be used in the first reaction without limiting the overall number of targets, because the universal adapter system enables parallel amplification of multiple adapter-ligated products in the second stage.
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 efficient and specific amplification of multiple targets in a single reaction, reducing artifact formation and improving the yield and purity of target-specific amplicons, thereby enhancing the reliability of downstream sequencing and analysis applications.
Implementation Method 1
selective amplification is typically achieved through use of one or more primers that can selectively hybridize to, or selectively promote the amplification of, a particular target nucleic acid molecule
Implementation Method 2
amplifying a plurality of target sequences within a sample including two or more target sequences
Implementation Method 3
The formation of such artifacts in amplification reactions, even when only a single pair of primers is employed, can complicate downstream applications such as qPCR, cloning, gene expression analysis and sample preparation for next-generation sequencing
Data Source
AI summary
The present invention provides methods, compositions, kits, systems and apparatus that are useful for multiplex PCR of one or more nucleic acids present in a sample. In particular, various target-specific primers are provided that allow for the selective amplification of one or more target sequences. In one aspect, the invention relates to target-specific primers useful for the selective amplification of one or more target sequences associated with cancer or inherited disease. In some aspects, amplified target sequences obtained using the disclosed methods, kits, systems and apparatuses can be used in various downstream processes including nucleic acid sequencing and used to detect the presence of genetic variants.