NEXUS SeqFold Studio · Assay design research

qPCR & ddPCR Primer / Probe Designer

Generate research-stage PCR primer pairs and internal hydrolysis-probe candidates from DNA/cDNA, inspect predicted sequence metrics and potential secondary-structure risks, and export your assay designs. Computation stays on your device.

qPCR / RT-qPCRDroplet digital PCRUnmodified hydrolysis-probe candidatesLocal FASTA off-target screeningCSV / JSON export

1 · Target sequence and assay preferences

Use a target DNA or cDNA region, with optional FASTA header. One record per run; A/C/G/T and unresolved N positions are supported. Oligos will not overlap N positions.

Enter your template, select assay settings, and run the designer.

Research use only: This is an original heuristic screening engine based on SeqFold's existing unmodified DNA Tm estimate. It is not Primer3, Primer-BLAST, a validated diagnostic assay, or a substitute for in vitro efficiency/specificity tests. It cannot predict modified-probe Tm, isoform specificity, genomic DNA exclusion, or clinical diagnostic performance.

2 · Ranked assay candidates

Ranked by Tm, composition, simple secondary-structure flags and probe placement—not empirical amplification performance.

Awaiting design
Designed primer pairs and probes will appear here.

3 · Local primer-pair specificity check

Paste a user-selected FASTA reference collection to screen the top-ranked primer pair for potential amplicons. This scans only the sequences provided, never NCBI's full genome database.

No local specificity analysis run.

Verify with NCBI Primer-BLAST ↗Primer3Plus / Primer3 resources ↗Bio-Rad ddPCR design guidance ↗
Before ordering, check reference sequence identity, off-targets, variants at primer/probe sites, primer/primer probe ΔG using independently validated tools, RT controls for RNA, efficiency/dynamic range for qPCR, droplet separation and rain for ddPCR, and positive/negative controls. A negative local FASTA result does not establish whole-genome specificity.

Related NEXUS tools

Primer specificity & validationGeneral PCR primer designqPCR / ddPCR QC analysisInstrument data importConnected Project Hub

All calculations run in this browser tab; no target sequence is transmitted to NEXUS by this tool. File export is local. Do not use patient-identifiable or restricted sequences on devices without appropriate privacy protections.