Asymmetric Targeted Amplification for Primer Dimer Control
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Solution Overview
Problem
Existing amplicon sequencing methods using multiplex PCR suffer from significant primer dimer formation and undesired mini-amplicon artifacts, leading to decreased efficiency and accuracy, requiring qPCR quantification and low base composition complexity sequences.
Innovation Solution
A targeted NGS library workflow using multiplexed PCR with overlapping primer pairs in a single tube, followed by indexing PCR, prevents primer dimer and mini-amplicon amplification by incorporating universal and indexing primers with specific sequences, allowing for contiguous coverage over target regions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiplex PCR is used to amplify multiple target regions in a single tube, then productivity and cost savings are improved, but primer dimer formation and undesired mini-amplicon artifacts increase
Solution Approach 1:
The patent introduces asymmetry in primer design by adding a universal sequence to only one primer of each target-specific primer pair. This asymmetric configuration prevents the formation of symmetric primer dimers while maintaining the ability to amplify multiple targets in a single tube, thus resolving the contradiction between high multiplexing capacity and primer dimer formation.
Solution Approach 2:
The universal sequence incorporated into one primer of each pair serves multiple functions: it enables the primer to bind to a common site across different targets, facilitates contiguous coverage through overlapping primer pairs, and prevents primer dimer formation by eliminating symmetry. This multi-functionality allows the system to maintain high productivity while avoiding harmful artifacts.
2Manufacturing precision
If overlapping primer pairs are used for contiguous coverage, then manufacturing precision is improved, but undesired mini-amplicon artifacts increase
Solution Approach 1:
By making the primer pairs asymmetric through the addition of a universal sequence to only one primer, the patent prevents the formation of mini-amplicons that would otherwise result from overlapping primer pairs. The asymmetry disrupts the symmetric binding required for mini-amplicon formation while preserving the overlapping design for contiguous coverage.
Solution Approach 2:
The universal sequence is strategically placed in one primer of each pair, creating a local quality difference that prevents mini-amplicon formation at overlap regions while maintaining contiguous coverage through the overlapping design. This localized modification resolves the contradiction between precision and artifact formation.
3Object-generated harmful factors
If prior art methods are used to address primer dimer artifacts, then harmful factors are reduced, but device complexity and process steps increase
Solution Approach 1:
The patent applies preliminary action by incorporating the universal sequence into the primer design before the PCR reaction begins. This pre-configured asymmetry prevents primer dimer formation from the outset, eliminating the need for subsequent qPCR quantification or other corrective steps, thus reducing both harmful factors and process complexity.
Solution Approach 2:
The patent extracts the problematic symmetric element from the primer design by making one primer in each pair asymmetric through the addition of a universal sequence. This extraction of symmetry eliminates the root cause of primer dimer formation without requiring additional process steps or complex equipment.
Data Source
AI summary
The present disclosure provides kits and methods for a targeted NGS library workflow comprising a multiplexed PCR for target amplification followed by an indexing PCR for incorporation of NGS adapter sequences. This workflow enables overlapping primer pairs in a single tube for contiguous coverage over target regions, while simultaneously preventing amplification of both primer dimers and undesirable mini-amplicons that result from overlapping primer pairs.