FILE SP–2026–08

ENGINE Primer3-py + SantaLucia

CLASS. TECHNICAL · PUBLIC

MODES 3 chemistries · simplex and multiplex

REPOSITORY persistent

07 Design · primers

SnapPrime

It designs primers in standard, probe and HRM mode, simplex and multiplex, with full thermodynamics and validated specificity. Minutes per target, where manual design costs hours.

And what is validated is not lost: every pair that clears specificity promotes itself into the institutional repository, with its own identifier and a life independent of the project that created it.

How to read this page

The thermodynamic thresholds are written down and are the ones the engine applies. A candidate does not carry an overall mark: it carries its seven dimensions in plain sight.

Tm 57–63 °C · GC 40–60 % · 18–25 pb
HX-PR-NNNNNN · persistent repository

weighted dimensions in the score, visible one by one: not an overall mark

8 pb

of exact match required at the 3′ end: the biochemical rule that decides whether it extends

≥ 30 pb

of enforced separation between amplicons in multiplex, so they can be told apart by size

tiers in the institutional repository, with automatic promotion on validation

What makes it unique

Universal primer design by consensus: it builds the multiple alignment of every target, profiles conservation position by position, excludes only clusters of three or more consecutive variable positions — tolerating synonymous variation at the third codon base — and validates with the biochemical extension rule: exact match over the last eight bases of the 3′ end.

A single pair that amplifies every target and is checked against each one, instead of one pair per target that then has to be made compatible. Neither Primer3 in its web interface, nor PrimerQuest, nor Beacon Designer combines intelligent consensus with biochemical extension validation.

01 The state of the art

Designing the primer is the easy part. What comes after is what costs.

Validating cross-dimers, checking specificity and making a multiplex compatible is where the hours go — and where combinatorial complexity pushes the laboratory into costly trial and error.

Tool

How far it goes

What SnapPrime adds

Primer3 in its web interface

Designs a correct pair over a sequence and is the field's de facto standard.

Uses Primer3-py as its engine and adds what is missing around it: taxonomic specificity, multiplexing and a persistent institutional repository — yesterday's design is still there, with its identifier.

PrimerQuest, Beacon Designer

Assisted design with per-chemistry templates and a polished commercial interface.

Hace consenso sobre alineamiento múltiple y aplica la regla de la semilla 3’ de 8 pb, y resuelve el multiplex por diseño en vez de por prueba y error.

Manual BLAST for specificity

Detects homologies against the public database and is every laboratory's trained reflex.

Evaluates behaviour, not similarity: SantaLucia NN thermodynamics, mismatches at the extending end and secondary structure, with Tm inside the 57-63 °C window.

A spreadsheet with the group's primers

It keeps what already worked and avoids redesigning from scratch.

Stores every pair with an identifier, a validation level and a record of what it was validated against — and survives a change of staff.

02 The modes and their rules

Three chemistries, and hard rules the engine will not let you skip.

In multiplex, the probe is mandatory: the engine validates it and the interface disables the rest. A hard rule prevents an assay that collapses in the laboratory.

Standard

Amplification with intercalating-dye detection

Forward and reverse pair with the wizard's parameters, meant for non-specific fluorescence reading or gel.

Simplex

Probe · FRET

Pair plus a labelled probe

The mandatory chemistry in multiplex: each target carries its probe, and that is what allows them to be told apart in the same tube.

Mandatory in multiplex, by hard rule of the engine

HRM

Short amplicon for a melting curve

Amplicons of 50 to 150 bp with melting-curve prediction through our own implementation of the Poland-Scheraga model.

Simplex · curve prediction included

Thermodynamic thresholds applied

57–63 °C

melting temperature, optimum at 60 °C, with control of the difference between the two primers

≥ −9,0

kcal/mol for homodimer and heterodimer: below that, the candidate is discarded

≥ −2,0

kcal/mol for hairpin, and ≥ −5.0 for 3′-end stability

40–60 %

GC content, with a length of 18 to 25 bases

03 How it works

From sequence to validated pair, with its file.

The score is not a mark: it is seven weighted dimensions you can inspect one by one to understand why one candidate falls below another.

Input and metadata resolution

Sequences imported from BioMiner or loaded as a file or accession number, with automatic resolution of the organism and its annotation.

Design in the chosen chemistry

Candidate generation with standardised parameters. In universal mode the multiple alignment of every target is built and design happens over the conserved region.

Multi-criteria scoring

Melting temperature and its differential, GC content, homodimers, hairpins, heterodimers and the specificity result: seven weighted dimensions, each of them inspectable.

Multiplex optimisation

Every possible heterodimer between the panel's pairs is precomputed and simulated annealing searches for the combination that minimises cross-dimers while also enforcing amplicon size separation.

Taxonomic specificity

Search against the project database and the taxonomic database, with analysis of mismatches at the 3′ end and a verdict that distinguishes unexpected amplification from mispriming.

Automatic promotion

Once specificity is cleared, the pair enters the institutional repository with its own identifier, an assigned tier and the record of what it was validated against. No manual step to forget.

04 The institutional repository

A validated primer is an asset of the laboratory, not of the project.

Primers survive the deletion of the project that created them. With their own identifier, their tier and the record of what they were validated against.

Gold

Validated with full specificity and breadth demonstrated over several organisms.

Silver

Validated, with a narrower scope or less accumulated evidence.

Bronze

A solid design with partial validation: it works, and how far it works is known.

Unvalidated

Designed but not through specificity. It appears flagged as such; it is neither hidden nor discarded.

Promotion is automatic: it happens as soon as specificity validation comes back green, with nobody having to remember to file it. And breadth counts — a pair that works for several organisms scores above one that works for a single one.

05 The contract

What goes in, what comes out, what it chains to.

Candidate regions from BioMiner with their traceability, targets recommended by the target advisor, or your own sequence as a file or public accession number.

Out

Ranked candidates with forward and reverse sequences, probe where applicable, melting temperature, GC content, full thermodynamic data, melting curves, the specificity result, and export to spreadsheet, report or synthesis order.

Chains to

To the in silico validator, which puts the pair through the full biophysical test, and from there to the kit generator. The repository feeds both without designing again.

06 Technical sheet

Chemistries

Standard, hydrolysis probe and high-resolution melting, in simplex; multiplex with probe only, by engine rule.

Universal by consensus

Multiple alignment, conservation profiling, exclusion of clusters of three or more consecutive variable positions with tolerance for synonymous variation at the third base, and cross-checking of exact forward and reverse-complement matches on every template.

Multiplex

Quadratic-order precomputation of heterodimers over the panel and optimisation by simulated annealing, with a minimum separation of 30 base pairs between amplicons.

Specificity

Percentage identity and coverage, mismatches over the last five bases, and a verdict separating unexpected amplification from mispriming.

Repository

A persistent identifier per primer, four tiers, a composite score from design, a tier multiplier and a breadth bonus, and a life independent of the originating project.

Export

Spreadsheet, PDF report and an oligonucleotide synthesis order ready for the supplier.

Limit · multiplex

Simulated annealing is not deterministic: the same input can give different combinations between runs. It is in the debt register, with a configurable seed as the planned fix.

Limit · HRM

Melting-curve prediction is our own implementation and has not yet been checked against the field's public references over a panel of sequences. It is used as guidance, not as a measurement.

Limit · databases

The taxonomic database for specificity is updated by hand. Automating that refresh is pending, and in the meantime it is worth checking its date before a campaign.

Design engine: Primer3-py. Nearest-neighbour thermodynamics: SantaLucia. Melting prediction for HRM: our own implementation of the Poland-Scheraga model. Multiplex optimisation: simulated annealing. Specificity: local similarity search against the project database and the taxonomic database. Strain-specific pipeline: minimap2 2.28, samtools 1.21, bedtools 2.31.1 and Snippy 4.6.0.

Minutes, not days. And with the why behind every rejection.

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