Site-saturation mutagenesis

1. 01Overlapping forward and reverse primer pairs carrying a degenerate codon at each chosen residue
2. 02One reaction condition for every site: the overlap melting temperature and the 3-prime melting temperature are matched across all chosen sites, which is what lets a single thermal profile amplify them together
3. 03Any degenerate codon, with NNK, NNS, NNN, NDT, VHG and DBK offered as presets and the real amino-acid coverage of each stated
4. 04Flanking primers defaulted from the live plasmid, offered only where each occurs exactly once, because the solver requires uniqueness
5. 05Every solver parameter exposed as a control: primer and overlap size ranges, 3-prime and 5-prime end sizes, GC bounds, temperature windows and step, and each score weight
6. 06Optional primer3 hairpin and dimer penalties folded into the score
7. 07A placement preview showing exactly where each oligo lands before anything is written to the record
8. 08Designed primers placed on the map at their real coordinates, and CSV export that expands every degenerate codon you asked for

The designer is a faithful port of Mutation Maker's SSM fast-approximation solver (Merck, GPL-3), running headless in packages/ports and validated byte-for-byte against the real library on the pEGFP-N1 record this app ships: 166 assertions covering the melting-temperature calculator, the overlap search, primer growth, scoring and the whole solve.

Everything runs client-side. Files are parsed in the browser and projects are saved on this machine, so an unpublished sequence is never uploaded.

What is the difference between this and multi-site mutagenesis?

Saturation mutagenesis randomises a residue to many amino acids at once using a degenerate codon, and designs an overlapping primer PAIR per site. Multi-site mutagenesis makes specific substitutions at several positions and designs ONE single-strand oligo per group of nearby sites. They answer different questions and neither replaces the other.

Why does it insist on flanking primers?

The 3-prime temperature window is derived from the melting temperatures of the flanking pair, so they are real inputs with real consequences. You can take the window over yourself instead, and the panel says so where that changes the design.

Which degenerate codon should I use?

NNK and NNS reach all 20 amino acids with one stop codon in 32; NDT reaches 12 with no stop, which is often preferable when stops would waste library capacity. The tool states the real coverage of each rather than assuming NNK.