Primer Design

Design PCR primers for Brassica napus reference genomes using Primer3, with per-primer genomic exact-match specificity evaluation by BLAST. Supported genomes: ZS11v1, Westarv1, Darmorv1.

1 Design Mode & Target Templates

Design Mode: Standard PCR (Genomic + Flank) qPCR (Transcript/CDS) Custom DNA Sequence

Input Model: Gene ID TE ID Custom DNA Sequence

Gene IDs (Up to 30)

Flank Size (bp, each side)

Template = gene region ± flank from reference genome coordinates.

2 Design Parameters

Reference Genome

Product Min (bp)

Product Max (bp)

Return Primer Pairs

Advanced Options (Thermodynamic Constraints, Penalties, and Specificity)

Strict Mode Preset

Preset will update product size range and key thermodynamic constraints.

Template masking before primer design (DUST) Use hard mask (N) instead of lowercase mask Enable internal oligo (hyb oligo) picking

Thermodynamic Constraints

Opt Size (bp)

Min Size (bp)

Max Size (bp)

GC Clamp (3' end)

Max Poly-X (nt)

Max 3' End Stability

Opt Tm (°C)

Min Tm (°C)

Max Tm (°C)

Opt GC (%)

Min GC (%)

Max GC (%)

Max Self Compl (Any)

Max Self Compl (3')

Pair Max Compl (Any)

Pair Max Compl (3')

Penalty Weights

Weight Tm < Opt

Weight Tm > Opt

Weight GC < Opt

Weight GC > Opt

Weight Self Compl (Any)

Weight Self Compl (3')

Weight Pair Tm Diff

Weight Pair Compl (3')

Specificity Evaluation (BLAST)

Max Mismatches per Hit

3' Perfect Match (bp)

Max Off-Target Amplicon (bp)

Candidate Evaluation Mode

Secondary Structure Risk (Hairpin / Dimer)

Enable structure-risk evaluation

Max Hairpin Tm (°C)

Max Homo-dimer Tm (°C)

Max Hetero-dimer Tm (°C)

Sequence Constraints (0-based Coordinates)

Targets (start,len; ...)

Excluded Regions (start,len; ...)

Included Region (start,len)

Pair OK Regions (ls,ll,rs,rl; ...)

Overlap Junctions (positions)

Force Left/Right Position (0-based)

Force Left Start

Force Left End

Force Right Start

Force Right End

Reaction Chemistry & Tm Model

Monovalent Cations (mM)

Divalent Cations (mM)

dNTP Concentration (mM)

Primer Concentration (nM)

Annealing Temp (°C)

Tm Formula

Salt Correction

Primer Candidates i Interpretation notes:
1) Batch mode may use accelerated specificity settings to improve web responsiveness.
2) DUST masking detects low-complexity segments, but is not a full repeat/TE mask.
3) For top candidates in repetitive regions, rerun as single-template or use orthogonal validation.

Submit the form to generate primer candidates.