RT-qPCR Primer Design (Bio-chat Series)
This skill provides a professional-grade workflow for designing high-specificity RT-qPCR primers and probes, optimized for complex biological samples.
Key Capabilities
- Homology-based Junction Mapping: Uses multiple anchor points and sliding-window alignment to map exon-exon junctions from a well-annotated ortholog (e.g., Human NM_) to a predicted target transcript (e.g., Vero XM_). This ensures gDNA-safe design even when target annotations are missing.
- High-GC Template Optimization: Implements the full SantaLucia (1998) Nearest-Neighbor thermodynamic model for precise Tm calculation, essential for templates with >70% GC content (e.g., Pseudorabies virus).
- TaqMan & SYBR Green Support:
- TaqMan: Automatically designs probes with Tm 8-10°C higher than primers, following standard real-time PCR design rules (no 5' G, no poly-G runs).
- SYBR: Strict 3' quality control and secondary structure screening to minimize primer-dimers.
- Off-target Defense: Supports an optional
--offtarget transcript to check for 3' end mispriming, ensuring high specificity in mixed cDNA samples.
Workflow
- Target Identification: Provide a NCBI Accession (NM_/XM_) or a local FASTA sequence.
- Homology Mapping (Optional): For predicted monkey transcripts, provide a Human ortholog Accession to activate "Hardcore Mode" for junction mapping.
- Automated Design: Execute
scripts/design_qpcr_assay.py with specific parameters:
--target: Target accession (e.g., NM_001256799)--homolog: Human ortholog for mapping (e.g., NM_001256799 for a monkey gene)--offtarget: Accession for 3' specificity check (e.g., a homologous gene in the same family)
- Validation: Review the top-scoring sets based on Tm, GC, 3' quality, and gDNA safety.
Technical Standards
- Tm Calculation: SantaLucia (1998) Nearest-Neighbor (NN) model with salt and terminal correction.
- gDNA Defense: Primers are required to span exon-exon junctions by at least 5bp on each side for stability.
- 3' Quality: Strict GC-clamp control and mismatch prevention.
Designed by ZJU PhD @ Bio-chat Community