---
title: "qPCR primer and probe designer"
method: POST
path: "/qpcr_assay_design"
tags: ["Primers & oligos"]
---

# qPCR primer and probe designer

`POST /qpcr_assay_design`

## Request

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

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.

## Request body

- object
  - `template` string, required: Nucleotide sequence (raw or FASTA; IUPAC accepted).
  - `ampliconMin` integer: Shortest product (bp). qPCR wants a short amplicon so every cycle doubles.
  - `ampliconMax` integer: Longest product (bp). Past ~200 bp the efficiency starts to fall.
  - `tmMin` number
  - `tmOpt` number
  - `tmMax` number
  - `tmMaxDiff` number: Max Tm difference within a pair (°C). Tighter than ordinary PCR.
  - `gcMin` number
  - `gcMax` number
  - `targetStart` integer: 1-based inclusive start of a region the product must span (optional).
  - `targetEnd` integer: 1-based inclusive end of the target region (optional).
  - `maxReturn` integer: Number of best assays to return.
  - `oligoNM` number: Total strand concentration (nM).
  - `naMM` number: Monovalent cation [Na+]/[K+] (mM).
  - `mgMM` number: Divalent cation [Mg2+] (mM).
  - `dntpMM` number: Total [dNTP] (mM), chelates Mg2+.

## Response `200`

Tool ran successfully.

- SuccessResponse: Standard success envelope returned by every single-tool call.
  - `ok` unknown, required
  - `tool` string, required: The tool slug that ran.
  - `result` object, required: Tool-specific output object. Its shape depends on the tool — call `GET /{tool}` or see the docs for each tool's fields.
  - `gate` GateResult
    - `pass` boolean, required: True if and only if every hard check passed.
    - `checks` GateCheck[], required
      - `id` string, required
      - `label` string, required
      - `severity` 'hard' | 'soft', required
      - `pass` boolean, required
      - `message` string, required
      - `value` number
      - `threshold` string
    - `notChecked` string[], required: Honest list of what this gate does NOT verify.
  - `provenance` Provenance, required
    - `apiVersion` string, required
    - `tool` union, required: Tool slug, or an array of slugs (one per step) for a workflow.
      - string
      - string[]
    - `generatedAt` string, date-time, required: ISO 8601 timestamp of when the result was generated.

## Other responses

- `400`: Invalid argument or the tool could not run.
- `404`: Unknown tool.
- `429`: Rate limited (metered tools only). Body carries code `rate_limited` and retryable `true`; honor the Retry-After header.
- `503`: A metered tool is momentarily at capacity. The identical request should succeed after Retry-After; body carries code `upstream_unavailable`.

---

[API](https://skmtc.dev/seqbench/apis/seqbench-api.md) · [All operations](https://skmtc.dev/seqbench/apis/seqbench-api/llms.txt) · [OpenAPI document](https://skmtc.dev/seqbench/apis/seqbench-api/revisions/132575216865?raw)