Quality Analysis Results
How the Quality Score is Calculated
This analyzer assesses multiple essential biophysical characteristics of PCR primers to calculate an overall quality score out of 100:
1. Length: Evaluates if the primer length is within the ideal 18–25 bp range. Points are deducted for primers under 15 bp or over 30 bp.
2. Melting Temperature (Tm): Calculated using the highly accurate SantaLucia Nearest-Neighbor thermodynamic values (with standard 50mM Salt, 250nM primer). Penalties apply if Tm is outside the optimal 55–62°C range.
3. GC Content: Validates GC% against the ideal 40–60% window to avoid weak amplification or high Tm values.
4. 3' End GC Clamp: Having 1 to 2 G/C bases at the 3' end secures the polymerase binding. Missing clamp or having too many G/C bases (causing GC runoff) triggers penalties.
5. Base Runs: Identifies long repeats of identical bases (4+ contiguous A, T, G, C) which lead to polymerase slippage.
6. Dimer and Hairpin Risks: Uses a sliding hybridization alignment to find self-complementarity of 4+ bases, especially at the 3' end, which causes primer-dimer formation.
How to Optimize Suboptimal Primers
If your primer quality score is below 80, consult the Actionable Redesign Tips output widget. Common optimization remedies include:
- If Tm is too low: Extend the primer length by 2-3 bases at the 5' or 3' end, or shift the primer to a GC-rich region.
- If there is high dimer risk: Shift the primer binding site by a few bases along the template sequence to break up self-complementary zones.
- If no GC clamp is present: Try shifting the 3' end of the primer to terminate with a G or C.