Phase 9 · Experimental validation planning

Variant-aware Primer Design

Turn an exact sequence variant into experimental primer candidates: flanking PCR/Sanger pairs, SNV allele-specific PCR, indel junction assays and paired WT/ALT hydrolysis-probe sequences.

Research scope: Phase 9 starts from a validated local DNA window and exact REF/ALT allele. Primer3 and secondary-structure metrics are used for candidate generation. Allele specificity, genome-wide specificity, analytical sensitivity and diagnostic performance are not established in silico.

1. Variant & sequence context

SourceManual entry

Gene—

Genomic record—

Variant—

Variant position in sequence window (1-based)

For an insertion, this is the insertion boundary position supplied by the genomic handoff.

Reference allele (REF)

Use an empty field for a pure insertion.

Alternate allele (ALT)

Use an empty field for a pure deletion.

Design mode

DNA sequence window

Exact genomic handoff is preferred because Phase 9 verifies the REF allele against this sequence before designing primers.

Open Phase 9 from the Genomic DNA validation panel, or enter a validated sequence window manually.

2. Design parameters

Minimum product size (bp)

Maximum product size (bp)

Primer pairs/candidates

Sanger sequencing margin (bp)

Avoids placing flanking primers too close to the variant.

Tm min / opt / max (°C)

GC min / max (%)

Optional ARMS extra mismatch Generate a heuristic penultimate mismatch suggestion

Off by default. This is an experimental optimization suggestion, not a specificity guarantee.

Ready.

3. Assay summary

Variant type—

Flanking pairs0

Allele-specific0

Junction pairs0

Probe pairs0

4. Flanking PCR / Sanger candidates

These pairs place the variant inside the amplicon and keep primer binding sites away from the variant according to the sequencing margin.

Rank	Forward primer	Reverse primer	Tm F/R	GC F/R	Ref / Alt amplicon	Variant position	Heterodimer	Score

5. SNV allele-specific PCR candidates

WT and ALT primers differ at the 3′ allele-discriminating base and share a common opposite primer. Candidate ranking is sequence-based; annealing temperature and allele discrimination must be optimized experimentally.

Rank	Orientation	WT-specific primer	ALT-specific primer	Common primer	Tm WT/ALT/common	Amplicon	Score	Optional mismatch

6. Indel junction-specific candidates

Junction primers span the changed sequence boundary and are retained only when the exact primer sequence is absent from the opposite allele sequence.

Rank	Allele	Junction primer	Orientation	Common primer	Tm J/common	Amplicon	Heterodimer	Score

7. WT / ALT hydrolysis-probe sequence candidates

The SNV is placed internally in paired WT/ALT probe sequences. No fluorophore, quencher, MGB/LNA chemistry or reaction condition is selected.

Rank	WT probe	ALT probe	Tm WT/ALT	GC WT/ALT	Variant in probe	Score