Phase 12 · Assay QC & specificity

Primer Pair QC → Target Validation → Specificity Screen

Review a candidate primer pair before wet-lab use: sequence QC, Tm balance, 3′ complementarity, exact intended-amplicon validation, user-supplied reference-panel screening and optional NCBI BLAST screening.

Research scope: this phase is a screening layer. It does not establish assay sensitivity, efficiency, diagnostic specificity or clinical validity. Remote BLAST is intentionally user-triggered and rate-limited.

1. Primer pair

Candidate from Variant-aware Primers

You can enter a pair manually or arrive here from Phase 9.

Primer A (5′→3′)

Primer B (5′→3′)

Minimum amplicon

Maximum amplicon

Organism for optional BLAST

Ready.

2. Intended target sequence

Validated DNA target / genomic window (optional but strongly recommended)

Exact pair-level amplicons are located in either primer orientation. If more than one exact amplicon is found, intended-target uniqueness is not established.

3. Reference-panel specificity

User-supplied FASTA panel (optional)

Designed for a compact reference/off-target panel. Total input is capped at 500,000 nt; this is not a substitute for whole-genome search.

4. QC summary

Pair score—

Verdict—

ΔTm—

Target amplicons—

Panel amplicons—

Primer A

Primer B

Pair checks

Intended target

Reference-panel hits

5. Optional NCBI BLAST specificity screen

Why optional? NCBI BLAST is an external public service. Phase 12 submits each primer separately only when you request it, then combines compatible same-accession/opposite-strand hits as approximate pair-level off-target candidates.

Not started. After submission, wait at least 10 seconds between checks.

Accession	Approx. amplicon	Forward hit	Reverse hit	Reference title