Multiplex PCR Primer Design Service (Multiplex PCR / Tiling Amplicon)

Product Introduction
Based on the advanced PrimalScheme3 algorithm framework, this service provides an efficient and stable multiplex PCR primer design solution.

By combining tiling amplicon design, overlap strategy, and primer pool optimization, it enables continuous coverage of target sequences and high-success amplification. The service is suitable for pathogen detection, genotyping, high-throughput sequencing library preparation, and related molecular testing applications.

Core Advantages

1. Strong Continuous Coverage Through Overlap Design

Adjacent amplicons are designed with rational overlap regions, typically 10-50 bp, to improve sequence continuity.

Key benefits:

• Reduces the risk of coverage gaps caused by amplicon failure
• Improves data completeness
• Enhances overall assay stability

2. Optimized Multiplex PCR Pooling

The system automatically groups primers into optimized multiplex pools. Depending on project requirements, 2-4 pools can be selected.

Key benefits:

• Reduces primer-dimer formation
• Minimizes non-specific amplification
• Improves multiplex amplification success rate
• Supports more stable amplification across multiple targets

3. Intelligent Primer Screening for High Specificity

Primer candidates are screened through multiple design parameters to improve specificity and amplification reliability.

Evaluation factors include:

• Tm consistency
• GC content
• Primer-dimer risk
• Hairpin or secondary structure risk
• Compatibility between primer pairs

Optimization goals:

• Automatically optimize primer combinations
• Improve amplification specificity
• Ensure a stable and reliable PCR system

4. Tiling Amplicon Strategy for Full-Coverage Design

The target sequence is divided into multiple amplicon regions to achieve continuous and uniform coverage.

Design features:

• Segmental amplicon design for continuous target coverage
• Supports long sequences
• Supports highly variable regions
• Supports MSA-based design for variable targets
• Provides uniform coverage suitable for sequencing applications

5. Sequencing Adapter-Compatible Design

Sequencing adapters can be directly fused to the 5' end of primers, enabling integrated amplification and library preparation.

Supported platforms:

• Illumina
• MGI

Supported workflow modes:

• One-step PCR: direct adapter-linked amplification
• Two-step PCR: universal adapter plus index amplification

Automatic optimization includes:

• Balancing adapter and primer Tm
• Avoiding adapter-induced secondary structures
• Controlling amplification length deviation

Result:

• Amplification and library preparation can be completed in one integrated workflow
• Experimental cost and time are significantly reduced

6. Highly Controllable Design Parameters

The design process supports flexible parameter adjustment according to different experimental needs.

Configurable parameters include:

• Amplicon length: 100-2000 bp
• Overlap length
• Primer Tm range
• Number of primer pools, also known as multiplex level
• High-GC region optimization

Key benefits:

• Adapts to different sequencing requirements
• Supports customized assay design
• Improves flexibility for complex target regions

7. Complete Output Ready for Experimental Use

The service provides complete deliverables that can be directly used for downstream experiments.

Deliverables may include:

• Primer sequences
• Optional adapter-containing primer versions
• Amplicon positions
• Pool grouping information
• Primer design parameters
• Coverage information
• Experimental-ready design results

Result:

• Users can move directly from primer design to experimental validation
• Reduces manual adjustment workload
• Improves project efficiency and reliability