Primer sequence (5′→3′)

Replace U with T Pre-aligned input: evaluate full length (3′ side follows the selection on the right)

Template sequence (5′→3′)

Compare against the reverse complement (5′→3′) 3′ end location:

By default, the rightmost (3′) 20 nt are evaluated for unaligned inputs. With “Pre-aligned”, the full length is evaluated and the 3′ side follows the selection above.

Position-weighted fit score

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Total effective weight: – Weighted mismatch sum: –

pos	w	primer	template*	d	w×d

* The template is reverse-complemented depending on settings, then evaluated on the rightmost 20 nt or the full length.
Method key points: IUPAC letters match by consensus-set overlap (intersection ≠ ∅ ⇒ match = 0, else mismatch = 1). Primer-side N always matches (0); blank on the template side counts as mismatch (1).
Score: Score = 100 × ( 1 − (Σ w_i d_i) / (Σ w_i) )

Method summary (for manuscript Methods)

This tool returns a normalized fit score (0–100) between a primer and a template over 20 nt (or the full aligned length), using position weights to emphasize the 3′ end (positions 1–12: 0.2; 13–16: 0.5; 17–20: 1.0). At each position, the mismatch indicator d_i is 0 for a match and 1 for a mismatch. IUPAC ambiguity codes are treated as consensus sets; a match occurs if the intersection of the primer and template sets is non-empty. The primer-side N always matches; blanks in the template are treated as mismatches. The rationale of emphasizing the 3′ end and using a weighted objective is consistent with primer design practice.