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Reference genome

Primer settings

Allele-specific PCR (AS-PCR / ARMS)

This mode designs two allele-specific reactions: WT-specific and MUT-specific. By default, primers are designed so that the allele-discriminating base is at the 3′ end of the allele-specific primer (no extra intentional mismatch).

Via genomic position
?
  • Format/Info:
  • Positions must correspond to the selected reference genome.
  • For SNVs, the position refers to the exact location of the variant.
  • For InDels, the two positions refer to the start and end position of the sequence region that is substituted with the given variant sequence.
  • Inserted bases can be specified either as a base sequence (e.g., AGT) or as the number of inserted bases (e.g., 3)
  • Allowed format: ChrY:2655470
  • Optional: upload a VCF (same chromosome as your variant) to spike additional variants into the reference sequence before primer design (patient-specific background).

Genomic position

SNV
InDel

Optional: VCF (same chromosome)

Variants from the file are applied to the reference window (gnomAD-style MAF not required). Your SNV/InDel above remains the primary design variant.

Via transcript-ID
?
  • Format/Info:
  • Positions must correspond to the selected reference genome and sequence type.
  • For SNVs, the position refers to the exact location of the variant.
  • For InDels, the two positions refer to the start and end position of the sequence region that is substituted with the given variant sequence.
  • Inserted bases can be specified either as a base sequence (e.g., AGT) or as the number of inserted bases (e.g., 3)

Transcript-ID

Reference

Relative position

SNV
InDel
?

Accepted formats:
CGACT[A>G]GC
CGACT[AC/-]GC
CGACT[-/TT]GC
CGACT[ACA/TT]GC


A substituted by G (SNV)
AC deleted
TT inserted
ACA substituted by TT (DelIns)

Via sequence input

Sequence


Primer parameters

These values are submitted with your design request. PCR / qPCR presets set target padding and the usual product size range (you can still edit afterward). Advanced keys override the built-in Primer3 defaults when set; leave Tm min/max blank to derive from optimal Tm ±2 °C.

Assay preset

PCR: target padding 50 bp per side, product size 400–800 bp. qPCR: padding 30 bp per side, product size 80–150 bp.

Core

Primer length

Primer Tm bounds (optional)

Leave blank to derive from optimal Tm ±2 °C. Otherwise overrides min/max.

GC limits

Ion & concentration

Complementarity & quality

Internal oligo (unused when internal oligo off)

Designing your primers

Finding optimal primer pairs for your variant.

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