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Algorithms

PrimerLab is built on top of rigorous, peer-reviewed bioinformatics algorithms.

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Primer Design Engine: Primer3

The core primer search is performed by Primer3, the industry standard for primer design.

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How it works

  1. Scanning: The sequence is scanned for all possible valid forward and reverse oligos based on size and Tm constraints.
  2. Filtering: Oligos failing basic QC (GC clamp, Ns, low complexity) are discarded.
  3. Pairing: Valid forward and reverse primers are paired to check if they generate an amplicon of the correct size.
  4. Ranking: Pairs are ranked using the penalty method (see Scoring).

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Thermodynamics: ViennaRNA

For secondary structure prediction (hairpins, dimers), PrimerLab uses the nearest-neighbor thermodynamic model provided by ViennaRNA.

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Why use thermodynamics?

Simple string matching (e.g., counting matching bases) is insufficient for predicting DNA hybridization. Thermodynamics calculates the Gibbs Free Energy (ΔG), which predicts the stability of a structure.
  • Formula: ΔG = ΔH - TΔS
  • Interpretation: A negative ΔG means the structure forms spontaneously. The more negative, the more stable (and problematic for primers).

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Specificity: BLAST+

For off-target checking, we use NCBI BLAST+.
  1. Database: A local BLAST database is created from your reference genome.
  2. Query: Primer sequences are blasted against this database using blastn-short task (optimized for short sequences).
  3. Filtering: Hits are filtered for significant 3’ end matches (the last 5-7 bases). A primer binding perfectly at the 3’ end to an off-target site is considered a high-risk hit.