Multiplex PCR Primers with Cleavable Adapters
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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, which can dominate reactions and complicate downstream applications like next-generation sequencing.
Innovation Solution
The use of target-specific primers with cleavable groups and adapters that are substantially non-complementary to each other, allowing for selective amplification and subsequent processing to reduce artifact formation, along with strategies like adapter ligation and reamplification to produce high-quality amplicons for downstream analysis.
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 nucleic acid amplification is improved, but the formation of non-specific 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 allows the first reaction to generate sufficient template material without worrying about artifact dominance, while the second reaction selectively amplifies only the intended targets, thereby resolving the contradiction between high productivity and artifact formation.
2Productivity
If conventional primers are used in multiplex PCR, then the amplification of multiple targets is achieved, but the selective amplification is complicated by primer-dimer formation that consumes critical reagents
Solution Approach 1:
The patent introduces an adapter sequence as an intermediary element that bridges the first and second amplification reactions. The adapter is ligated to the amplicons in the first reaction and serves as a recognition site for primers in the second reaction. This intermediary mechanism allows selective amplification of target sequences without cross-reactivity or primer-dimer formation between different target-specific primers, thereby preventing consumption of critical reagents by non-specific artifacts.
3Measurement precision
If selective amplification of specific targets is performed, then the specificity of amplification is improved, but the process is complicated by the formation of superamplicons that act as templates for extension
Solution Approach 1:
The patent performs preliminary ligation of adapter sequences to the amplicons generated in the first amplification reaction before the second amplification step. This preliminary action ensures that only amplicons with the correct adapter sequences (which correspond to the intended target sequences) can serve as templates in the second reaction. Superamplicons formed in the first reaction lack the proper adapter configuration and therefore cannot be extended by the target-specific primers in the second reaction, effectively preventing their propagation.
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 efficient amplification of dozens to thousands of target sequences in a single reaction with reduced non-specific products, improving the yield and quality of amplicons for applications like next-generation sequencing and disease diagnosis.
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 one or more target sequences using primers having a cleavable group
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.