Pipeline Configuration
Fine-tune every parameter of the 24-step primer design engine to match your experimental requirements.
Design Modes
Standard PCR
Default mode for routine primer design. Optimised for Taq and proofreading polymerases with standard cycling conditions.
Bisulfite (MSP)
Methylation-specific PCR. Accounts for C-to-T conversion, avoids CpG dinucleotides in primer 3′ ends, and adjusts Tm calculations for reduced sequence complexity.
Multiplex
Designs compatible primer pairs for multi-target panels. Cross-dimer checks enforced across all pairs with strict ΔG > −5.0 kcal/mol threshold.
Primer Parameters
- Optimal TmTarget melting temperature for primers (default: 62°C)Range: 58–65°C | ΔTm ≤ 1.5°C enforced
- Amplicon SizeDesired product length rangeDefault: 100–400 bp | Max: 5,000 bp
- Primer LengthNucleotide length of each primerRange: 18–25 nt | 3′ GC clamp required
- GC ContentPercentage of G/C bases in the primerRange: 40–60%
- Salt & Buffer ConditionsMonovalent cation concentration for Tm calculation (SantaLucia 1998 NN model)Default: 50 mM Na² 1.5 mM Mg² 250 nM primer
Quality Thresholds
- Hairpin ΔGMinimum free energy for hairpin formation — less stable is betterThreshold: > −2.0 kcal/mol
- Self-Dimer ΔGMinimum free energy for primer self-dimerisationThreshold: > −8.0 kcal/mol
- Cross-Dimer ΔGMinimum free energy for cross-dimer between forward and reverse primersThreshold: > −8.0 kcal/mol
- BLAST Off-TargetsMaximum allowed off-target matches in the reference genomeStrict: ≤ 2 mismatches in last 5 bp
Advanced Options
- Probe Design (TaqMan)Dual-labelled hydrolysis probe with 5′ reporter and 3′ quencher. Tm 68–70°C, 15–25 nt, no G at 5′ end.
- NGS Adapter TailingAutomatically adds 5′ overhang sequences for Illumina Nextera, TruSeq, or Ion Torrent library prep.
- SNP FilteringCross-references primer positions against dbSNP to avoid common variants that may affect binding.
- Repeat MaskingFilters primers overlapping with Dfam repeat elements and low-complexity regions.