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v0.4

Primer, Probe & Plasmid Design

Changes (6)
  • Probes can now declare a backbone modification (unmodified DNA, LNA, PNA, 2'-O-methyl). The declaration changes what is graded and what is only reported, rather than acting as a label
  • LNA: declare which positions will be LNA residues and the longest consecutive run is graded, because runs over four lower synthesis yield and purity (You et al. 2006, design rule iv). Graded acceptable at worst, since the cost is synthesis quality rather than assay failure
  • Known variant positions for probes: a variant falling under the probe within 1-2 bases of either end is graded a concern, because discrimination is significantly decreased there; nearer the centre is preferred (You et al. 2006, design rule v)
  • LNA advisory rules now open in place: triplet placement centred on the mismatch, the G-T mismatch exception, the modify-all-adenines strategy when the mismatch is unknown, and the warning that LNA-LNA base pairs make self-structure more of a problem than the unmodified-DNA screening here suggests
  • PNA states its boundary: it cannot act as a PCR primer, which is the basis of PNA clamping (Orum et al. 1993), and no Tm is reported because the PNA-specific formula is not implemented
  • The probe-to-primer Tm rule now shows a published 7-10 C recommendation beside its own unverified 5-10 C band instead of merging them (You et al. 2006)
v0.3

Primer, Probe & Plasmid Design

Changes (4)
  • MGB and molecular-beacon probes are no longer graded against hydrolysis-probe bands. The Tm gap over the primers and the GC content are shown for both, but marked not evaluated with the reason: the computed Tm leaves out MGB stabilisation (Kutyavin et al. 2000), and a beacon's opening depends on target binding competing with its stem (Bonnet et al. 1999; Tsourkas et al. 2003)
  • Known variant positions for primers: enter SNP positions on the target and each primer is checked for variants under it; one within 5 nt of the 3' end is a concern, a single one elsewhere is acceptable, two or more are a concern (Lefever et al. 2013; Stadhouders et al. 2010). Works in design and in evaluate mode
  • Two supplier conventions for hydrolysis and MGB probes: no run of four or more G, and prefer the strand with more C than G. Both are marked 'Evidence not yet verified' and can only lower a candidate to 'acceptable', never to 'concern'
  • Correction: the hydrolysis-probe GC band no longer cites Kutyavin et al. (2000), which does not state such a band; it is now marked 'Evidence not yet verified'
v0.2.1

Primer, Probe & Plasmid Design

Changes (4)
  • Faster design: primer and probe searches now run about four to five times faster with identical results, because windows outside the acceptable Tm band are dropped before structure screening and the dimer and repeat scans no longer allocate per position
  • Changing a primer length, amplicon or region setting now clears the previous result until you run again, as every other input already did, so a long template is no longer re-designed on each keystroke
  • CSV exports start with a UTF-8 byte-order mark, so spreadsheet apps show non-ASCII feature names correctly
  • The closing advisory section of a plasmid result now has its own heading instead of repeating 'Elements this version cannot detect'
v0.2

Primer, Probe & Plasmid Design

Changes (6)
  • Design impact warnings: while you set lengths, amplicon range, DMSO, primer Tm or Gibson fragments, a live panel lists every setting outside its recommended band and what it is likely to do to the experiment
  • Each primer pair, probe candidate and plasmid result opens with a design-impact summary; concerns are listed before acceptable deviations, and card headers show the count
  • Every warning restates its own rule's mechanism, carries that rule's evidence tier, and says 'Basis not yet verified' when the rule is unsourced; the full basis opens in place
  • Duplicate Golden Gate overhangs are now graded against Engler et al. (2008) instead of only being listed
  • Redesigned layout: tabs with a one-line description, one collapsible scope notice above the tabs, card-style input column and a full-width run button
  • Fixes: plasmid map arcs no longer paint as solid wedges; base-pairing alignments are readable in the light theme; rule labels keep their case (qPCR, Tm); wide tables scroll inside their card on phones; long plasmid names are shortened inside the map
v0.1

Primer, Probe & Plasmid Design

Changes (23)
  • Primer: Nearest-neighbour Tm from the SantaLucia (1998) unified parameter set, with the monovalent salt term, a von Ahsen (2001) monovalent-equivalent correction for Mg2+ and dNTP, and the DMSO term from the same paper
  • Primer: Candidate primer enumeration over a pasted target, with pair ranking by how many sourced bands each candidate sits inside
  • Primer: Hairpin, self-dimer and cross-dimer screening with the base-pairing alignment shown, and extra weight where the pairing reaches an extendable 3' end
  • Primer: Per-criterion evidence: every verdict opens to its recommended range, reason, evidence tier and citation (PMID / DOI), or states 'Evidence not yet verified' instead of inventing one
  • Primer: Conflicting recommendations are shown side by side rather than merged: the qPCR and endpoint amplicon windows, and the unpublished 3'-GC-clamp convention against the documented mispriming view
  • Primer: Evaluate-mode for primer pairs you already have, locating them on an optional target to size the amplicon
  • Primer: Advisory section for what the tool cannot decide, including genome specificity and exon-junction placement
  • Probe: Three probe chemistries kept apart, because their design constraints come from different mechanisms: hydrolysis (5'-nuclease), MGB-conjugated, and molecular beacon
  • Probe: Candidate enumeration on both strands with primer footprints excluded, so a probe is never proposed where it would compete with a primer
  • Probe: MGB length band cited to Kutyavin et al. (2000), which measured a 12-mer MGB probe matching the Tm of an unmodified 27-mer
  • Probe: Probe-to-primer Tm relationship graded only when a primer Tm is supplied, and flagged as an unverified band rather than presented as published
  • Probe: Beacon analysis of a sequence you supply: detects the stem it folds into, isolates the loop, and reports both Tm values
  • Probe: Stated capability boundary: beacon stem arms are not generated, because no citable rule set fixes the arm sequence
  • Plasmid: Annotation in precedence order: a GenBank FEATURES block is used as given, and inference only fills in what the record does not state
  • Plasmid: Every feature row carries how it was found — GenBank annotation, nucleotide motif, translated peptide, ORF scan, or computed — so a guess never looks like a record
  • Plasmid: Protein-level tag detection translates ORFs and matches standard epitope, affinity and protease-site peptides, which makes it codon-independent
  • Plasmid: Restriction site scan with circular wrap-around, unique-cutter summary, overhang classification, and Type IIS support
  • Plasmid: Circular and linear maps as inline SVG, with declared and inferred features drawn differently
  • Plasmid: Assembly checks: Gibson-style junction overlaps graded against Gibson et al., and Golden Gate internal-site and duplicate-overhang checks against Engler et al.
  • Plasmid: Validation checklist that leaves sequencing confirmation as a manual item rather than marking it passed
  • Plasmid: States what it cannot detect (CMV promoter, resistance cassettes, ColE1 origin, fluorescent proteins) instead of silently omitting them
  • Plasmid: Provenance panel declares that the enzyme table and feature library were not machine-verified in this build, because outbound access to REBASE was unavailable
  • Everything computed in the browser, in all three tabs; sequences are not uploaded