check_scorpion_stem
A Scorpion probe's two melting temperatures, side by side: the stem-loop's own hairpin and the duplex the loop makes with its target on the amplicon. A Scorpion is a primer carrying a stem-loop probe on a non-amplifiable blocker (Whitcombe 1999), and it reports by the loop finding its target and pulling the stem apart - so the design question is a RELATION between those two structures, not a property of either. Returns both temperatures, their difference, the stem's GC, and a verdict against WHATEVER margin you pass. It does not apply a margin of its own authority: the widely-quoted 5-10 °C figure comes from one design tool's tech note and has no primary citation, so the default is labelled as that rule of thumb and every result says which margin produced its verdict.
Input Schema
| Name | Required | Description | Default |
|---|---|---|---|
| probe | Yes | the whole probe element, 5' stem arm + loop + 3' stem arm. The element is what folds, so it is what the structure engine is given - the loop alone would compute a hairpin the molecule does not have. | |
| _files | No | The project: record references shaped like _subject. Their records become library documents a tool names in sequences/parts/document; a trace or alignment is found by file name. | |
| margin | No | how many °C the stem Tm must exceed the loop-target Tm for the verdict to pass. Yours to set: the 5-10 °C rule of thumb this defaults to the floor of is a design tool's, not a measured value from the primary literature. In °C. | |
| target | Yes | the loop's target on the amplicon, 5'->3'. This is the OTHER temperature: a Scorpion opens because the loop finds this, so it is the loop-target duplex that has to be stable, not the loop's own self-structure. | |
| _subject | No | The record to act on: exactly one of sequence, accession (+source), sample (an id from list_samples) or file ({name, text} or {name, base64}); record_index picks one record of several. | |
| conditions | No | salt, magnesium, dNTP and oligo concentrations plus the salt-correction method the stem Tm is measured in. Left unset it is filled from your reaction-conditions settings, and falls back to primer3's defaults when there are none. Advanced. | |
| stem_length | No | how many bases of the 5' end form the stem arm. Reported back rather than guessed. The two vendors disagree on the range - LGC Biosearch, who sell Scorpions, publish 5-7 nt and PremierBiosoft's design tool says 6-7 - so neither is enforced. In nt. |
Output Schema
| Name | Required | Description | Default |
|---|---|---|---|
| notes | Yes | why a temperature is missing, in the words the panel shows. Empty when both were computed | |
| stemTm | Yes | melting temperature of the probe element's own hairpin, °C - null when no stem forms | |
| probeTm | Yes | melting temperature of the loop-target duplex, °C | |
| difference | Yes | stemTm - probeTm, the quantity the design rule is about | |
| clearsMargin | Yes | whether the difference clears the margin GIVEN - null when either temperature could not be computed | |
| marginSource | Yes | where the margin came from, carried with the verdict so an unsourced rule of thumb is never mistaken for a measurement | |
| stemGcPercent | Yes | GC of the 5' stem arm - both vendors ask for a GC-rich stem |