Why did three substitutions produce only two oligos?

That is the point of the method. Sites close enough to sit under one primer are merged onto a shared oligo; only sites too far apart get their own. The solver searches every legal grouping and keeps the best-scoring one.

Why can the same request give different primers each time?

The upstream algorithm picks its mutagenic codons by random draw, so a request genuinely has many valid answers. This tool exposes the draw as a SEED, so a design is reproducible; re-rolling explores alternatives, and on some inputs every seed converges on the same answer, which the panel says in place.

What is the difference between this and saturation mutagenesis?

Multi-site mutagenesis makes the specific substitutions you name at several positions, with one single-strand oligo per group. Saturation mutagenesis randomises a residue to many amino acids using a degenerate codon and an overlapping primer PAIR. Different questions, different oligo geometry.

Cloning

Multi-site mutagenesis

Name the substitutions you want anywhere in a gene and get the mutagenic oligos for them, designed for the one-tube multi-site protocol. The work the tool does is the grouping: substitutions close enough to sit under a single primer are merged onto one oligo, sites too far apart get their own, and the solver searches every legal split of your sites rather than assuming one. Ask for three changes and you may well get two oligos, which is the method working, not a shortcut.

Open the editor Open the Site-directed mutagenesis sample

Five real substrates — EGFP, pBR322, an unannotated pUC19, a TEV-tagged vector and a linear genomic CDS: design SOE/QuikChange primers, or scrub sites silently

What it does

01One single-strand mutagenic oligo per GROUP of sites: substitutions close enough to share a primer are merged onto one oligo, and the solver searches every legal split of the requested sites for the cheapest set
02Several targets at one position collapse into a degenerate codon, so one oligo delivers the whole sub-library
03One reaction temperature for the whole set, reported with the design
04Amino-acid coverage stated as a percentage, so a design that cannot deliver everything you asked for says so rather than looking complete
05A design seed, so the same request reproduces the same oligos and a primer you ordered can be re-derived later
06Every solver parameter exposed: primer size and end sizes, GC bounds, temperature threshold range and step, codon usage threshold, and each score weight
07Codon usage from the bundled E. coli and S. cerevisiae tables, or from a live table fetched in the Codon Usage tool
08A placement preview showing exactly where each oligo lands before anything is written to the record
09Input validated before the run, with refusals that name the arithmetic rather than returning an empty table

What it runs on

The designer is a faithful port of Mutation Maker's QCLM workflow (Merck, GPL-3), running headless in packages/ports and validated against the real library on the pEGFP-N1 record this app ships: 63 assertions covering the melting-temperature calculator, both bundled codon-usage tables in full, the site-split search and the whole solve. The panel runs on @ports/enzymes, @ports/mutagenesis, @ports/oligo-structure, @ports/variant-effect.

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

Questions

Why did three substitutions produce only two oligos?: That is the point of the method. Sites close enough to sit under one primer are merged onto a shared oligo; only sites too far apart get their own. The solver searches every legal grouping and keeps the best-scoring one.
Why can the same request give different primers each time?: The upstream algorithm picks its mutagenic codons by random draw, so a request genuinely has many valid answers. This tool exposes the draw as a SEED, so a design is reproducible; re-rolling explores alternatives, and on some inputs every seed converges on the same answer, which the panel says in place.
What is the difference between this and saturation mutagenesis?: Multi-site mutagenesis makes the specific substitutions you name at several positions, with one single-strand oligo per group. Saturation mutagenesis randomises a residue to many amino acids using a degenerate codon and an overlapping primer PAIR. Different questions, different oligo geometry.

Also searched as

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