Primers & oligos

qPCR primer and probe designer

Design a qPCR assay: primer pairs under qPCR's own constraints (70-150 bp product, tightly matched Tm) each with the best hydrolysis (TaqMan-style) probe that fits between them. The probe sits 6-12 °C above the warmer primer, never begins with G — a 5' guanine quenches the reporter even after the probe is cleaved — carries no run of four identical bases, and is reported on whichever strand has more C than G, with that strand named. A pair whose window holds no legal probe is still returned in order, because a SYBR assay needs no probe and dropping it would answer the probe question when the primers were what was asked for. For an ordinary PCR product use primer_design, which has the wider amplicon range.

post/qpcr_assay_design

Request

  • Base URL: https://seqbench.com/api/v1
  • URL: https://seqbench.com/api/v1/qpcr_assay_design
  • Auth: none required

Request body

templatestring required

Nucleotide sequence (raw or FASTA; IUPAC accepted).

ampliconMininteger

Shortest product (bp). qPCR wants a short amplicon so every cycle doubles.

ampliconMaxinteger

Longest product (bp). Past ~200 bp the efficiency starts to fall.

tmMinnumber
tmOptnumber
tmMaxnumber
tmMaxDiffnumber

Max Tm difference within a pair (°C). Tighter than ordinary PCR.

gcMinnumber
gcMaxnumber
targetStartinteger

1-based inclusive start of a region the product must span (optional).

targetEndinteger

1-based inclusive end of the target region (optional).

maxReturninteger

Number of best assays to return.

oligoNMnumber

Total strand concentration (nM).

naMMnumber

Monovalent cation [Na+]/[K+] (mM).

mgMMnumber

Divalent cation [Mg2+] (mM).

dntpMMnumber

Total [dNTP] (mM), chelates Mg2+.

Response

Tool ran successfully.

okunknown required
toolstring required

The tool slug that ran.

resultobject required

Tool-specific output object. Its shape depends on the tool — call GET /{tool} or see the docs for each tool's fields.

Example response

{
  "provenance": {
    "apiVersion": "1.1.0"
  }
}

Changes

No recorded changes to this operation across all 1 revision of this API.