pcr_amplify
Amplify the subject template with a forward + reverse primer (pydna Anneal). Emits the first product as a NEW sequence record so downstream tools (digest, gel, map) operate on the amplicon, not the template — the tool→tool hand-off.
Input Schema
TableJSON Schema
| Name | Required | Description | Default |
|---|---|---|---|
| dmso | No | DMSO in the reaction, % (v/v) — depresses every primer Tm and therefore the annealing temperature. In %. Advanced. | |
| limit | No | min 3' footprint match length (pydna `limit`). In bp. Advanced. | |
| _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. | |
| forward | Yes | forward primer 5'→3'. | |
| reverse | Yes | reverse primer 5'→3'. | |
| _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. | |
| nn_table | No | nearest-neighbour table the Tm and Ta are computed on. Left unset it is the panel's own default, SantaLucia & Hicks 2004 unified. Values: DNA_NN1 (Breslauer 1986), DNA_NN2 (Sugimoto 1996), DNA_NN3 (Allawi & SantaLucia 1997), DNA_NN4 (SantaLucia & Hicks 2004 (unified)), RNA_NN1 (Freier 1986 (RNA/RNA)), RNA_NN2 (Xia 1998 (RNA/RNA)), RNA_NN3 (Chen 2012 (RNA/RNA, GU wobble)), R_DNA_NN1 (Sugimoto 1995 (RNA/DNA hybrid)). Advanced. | DNA_NN4 |
| conditions | No | the reaction buffer every temperature here is computed in - Na+/K+/Tris/Mg2+/dNTPs, the primer concentration, the salt-correction method and the melting model. Left unset it is filled from your reaction-conditions settings, so this node and the PCR panel describe ONE tube; with neither it falls back to pydna's Taq defaults, which is what every golden was captured under. Advanced. | |
| dmso_model | No | which published DMSO depression to apply; they disagree by 2 °C at 8% DMSO, so a Tm quoted without naming one is not reproducible. Values: ahs01 (Ahsen et al. (2001)), mus81 (Musielski et al. (1981)), cul76 (Cullen and Bick (1976)), esc80 (Escara and Hutton (1980)). Only applies when dmso is set. Advanced. | ahs01 |
| end_length | No | 3'-most bases that must pair exactly whatever the mismatch budget (QUEEN `endlength`) — a polymerase extends from a paired 3' terminus, so this is not negotiable by the budget. Advanced. | |
| mismatches | No | substitutions a primer may carry and still prime (QUEEN `{s<=N}`). 0 is pydna's exact search, which is what the panel opens on and what every golden was captured under. Advanced. | |
| inner_forward | No | nested PCR: the inner forward primer 5'→3'. With `inner_reverse`, `forward`/`reverse` are the outer pair and every output below describes ROUND 2. Advanced. | |
| inner_reverse | No | nested PCR: the inner reverse primer 5'→3'. Advanced. | |
| dimer_threshold | No | the dimer-free cut-off `primerDimerScore` is judged against (PCR-35). The default is the value PrimerROC publishes, which that paper fitted on its own gels and says is specific to its PCR conditions — refit it on your own gel-scored pairs with `primerRocFit` rather than guessing one. Advanced. |
Output Schema
TableJSON Schema
| Name | Required | Description | Default |
|---|---|---|---|
| record | Yes | the amplicon as a sequence record (subject for downstream nodes) | |
| sequence | Yes | amplicon sequence 5'→3'. Empty when the pair made no product | |
| conditions | Yes | the buffer every temperature here was measured in — salt, magnesium, dNTPs, oligo concentration, the salt-correction method and the NN table — or a note saying pydna's defaults were used | |
| forwardTmC | Yes | forward primer melting temperature, under the table and additives set above | |
| reverseTmC | Yes | reverse primer melting temperature | |
| productCount | Yes | number of products (>1 = non-specific pair) | |
| crossDimerTmC | Yes | the forward x reverse 3'-end cross-dimer temperature — the primer-dimer that extends into a band. 0 when no such dimer exists, and a value far below the 35 °C limit is still a real structure: this is the number to compare pairs on, because the boolean beside it only reports primer3's design cutoff | |
| annealingTempC | Yes | annealing temperature for the first product, under the Taq convention. Null when the pair made no product | |
| primerDimerRisk | Yes | true when the pair breaks one of primer3's own cross-dimer limits (35 °C at a 3' end, 45 °C anywhere) — branch a workflow on this rather than re-deriving it. FALSE IS NOT 'no dimer': those limits are primer3's design filter, and graded against 48 gel-tested pairs (Johnston et al., Sci Rep 9:209, 2019) they flag none of the 23 that produced a visible primer-primer band. Read `crossDimerTmC` beside it rather than branching on this alone | |
| primerDimerScore | Yes | PrimerDimer's score for the pair (Johnston et al., Sci Rep 9:209, 2019: one hetero- and both homodimers, the most negative kept, clipped at 0). Lower is more dimer-prone. On 48 gel-tested pairs it separates real primer-dimer bands better than the thal Tm beside it (AUC 0.842 against 0.727), and on the paper's own 319 gel-scored pairs it reads 0.961 against 0.750. Read `primerDimerVerdict` beside it for the published cut-off rather than inventing a threshold. Null when a primer carries a base other than A, C, G or T | |
| primerDimerCutoff | Yes | the dimer-free cut-off `primerDimerVerdict` was read against, so a turn never has to assume which threshold produced the verdict. -4.43 is the published value, fitted on the paper's own PCR conditions | |
| primerDimerVerdict | Yes | true when `primerDimerScore` falls BELOW PrimerROC's dimer-free cut-off, which is the paper's own verdict rather than primer3's design filter (Johnston et al., Sci Rep 9:209, 2019). The cut-off defaults to the published -4.43 and is the `dimerThreshold` input: the paper fitted it on its own gels and says so, so a lab with its own gel data refits it with `primerRocFit`. Branch on this for the dimer question and on `primerDimerRisk` for the would-primer3-reject-this question — they answer different things. Null when a primer carries a base other than A, C, G or T | |
| round1ProductCount | Yes | nested PCR only: how many products the OUTER pair made in round 1 (the first one is re-amplified). Absent for a single-round run |