Looping Amplification Primer Design for High-Level Multiplexing
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Solution Overview
Problem
Target re-sequencing in clinical research and disease diagnosis faces challenges with low target coverage, low sequencing specificity, and high costs due to limitations in increasing multiplexing levels beyond 10-plex, primarily due to primer dimer formation and reduced sequencing specificity.
Innovation Solution
A method involving the use of forward and reverse primers with target-specific and common sequences that form a stem-loop structure, allowing for the amplification of over 15,000 target nucleic acids per reaction in the Fluidigm ACCESS ARRAY system, utilizing on-chip barcoding and specific primer designs to enhance multiplexing without sacrificing sequencing specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiplexing level is increased beyond 10-plex, then more targets can be amplified per reaction, but primer dimer formation increases and sequencing specificity decreases
Solution Approach 1:
The primer is divided into distinct functional segments: a sample-specific portion for target recognition and a common sequence portion for amplification. This segmentation allows the sample-specific portion to be optimized for specificity while the common sequence enables efficient amplification, resolving the contradiction between multiplexing capacity and sequencing specificity
Solution Approach 2:
The common sequence portion serves multiple functions: it provides universal binding sites for amplification across all samples, enables standardized sequencing primer binding, and facilitates on-chip barcoding. This multi-functionality allows high multiplexing without sacrificing specificity, as the universal portion is designed to work consistently across all targets
2Productivity
If multiplexing level is increased, then more targets can be amplified per reaction, but cost decreases
Solution Approach 1:
Multiple functions are merged into the primer design: sample identification, target amplification, and sequencing preparation are all integrated into a single primer component. This merging eliminates the need for separate barcoding steps and custom sequencing primers, reducing overall cost while enabling high multiplexing
Solution Approach 2:
The common sequence portion is designed to be self-sufficient, providing all necessary binding sites and structural elements for amplification and sequencing without requiring additional reagents or components. This self-service design simplifies the workflow and reduces reagent costs, making high multiplexing economically viable
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 increases multiplexing levels while maintaining high sequencing specificity, enabling cost-effective and efficient targeted sequencing library preparation, allowing for the simultaneous amplification of thousands of targets with improved genomic coverage.
Implementation Method 1
a target nucleotide sequence is flanked by the common sequence on one end and its reverse complement on the other end, whereby a single strand of the target amplicon can form a stem loop structure
Data Source
AI summary
The present disclosure provides a “looping amplification” method to increase the specificity of nucleic acid amplification. This increased specificity facilitates multiplexing to a much higher degree than was previously possible.