Primer design¶

A PCR primer has to bind one place on the template, melt near its partner, and end on a base the polymerase can extend. You say how far each primer may move; the package judges every binding site in that room and keeps the best pair. One command does it and writes the sheet you order from.

When to use it¶

• You want every check on a pair before you order it.
• A primer has to carry a tail: a restriction site, a Type IIS site, or bases that hold a fusion in frame.
• You are amplifying genomic DNA, where a pair clean on a plasmid may prime in twenty places.

You do not run this for the cloning pages: Golden Gate, Gibson, restriction and ligation and Gateway design their own primers.

What you need¶

One record to amplify from, as SnapGene .dna, GenBank or FASTA, and the region you want; on a genome, that FASTA and its index. The run below uses a file that ships with the repo.

Run it¶

pixi run mbio primers design tests/data/pUC19.dna --region 20..340 --out plan/

It prints the pair, then the file it wrote:

pUC19: CGGTGAAAACCTCTGACA / AATCGCCTTGCAGCACAT, 321 bp, Ta 63 °C, checks warn
plan/primers.tsv

--region 20..340 counts from 1 and takes both ends in; every verb here reads a span that way. Told nothing more, the design pins each primer's 5' end to one end of the span and varies only its length, so the amplicon is the 321 bp you asked for. Tell it more and the primer may move: a tail to anchor it, a junction to read across, a flank to sit inside. It judges every site that room allows and keeps the best pair, so a warning left on a primer is one nothing nearby could clear.

Ta 63 °C is the temperature to anneal at. checks warn means nothing failed and something wants a look.

What it wrote¶

That name links to what the run wrote, published here unedited. The same record and region always give the same pair, so changing the design means changing the command: --target-tm moves the temperature aimed for, 62 °C, and --polymerase picks whose rules judge the pair, Q5.

Check the pair on a genome¶

Give it a genome FASTA and the assembly's name, and it designs against the whole genome:

pixi run mbio primers design GENOME.fa --assembly ce11 --region chrI:401..700 --out plan/

The region now names the sequence it lies on, as NAME:START..END. The forward primer goes in the left flank and the reverse in the right, so the amplicon covers the whole region; --flank says how far out either may sit. The best-ranked pairs go into one genome search, and the first making only the amplicon you asked for wins. Where none does, the primers that primed elsewhere are ruled out and it searches again, and after a fixed number of searches it hands back the best pair it saw, marked not specific.

The summary line gains the genome and the region in front; a second line follows it, with whether the pair is specific or how many other amplicons it makes, how many searches that took, and the worst of its checks. Beside the sheet it writes genome.tsv, a row for each amplicon on the genome: its place in the spelling you typed, its length, whether the pair or one primer alone makes it, the mismatches at each end, and whether it is yours.

A large FASTA gets a perfect-match screen: above a size limit a site with one mismatch goes unseen, and the report says so. ipcr runs the search. It comes from bioconda, not PyPI, so pixi install brings it and a pip install does not.

No genome FASTA ships here and none is downloaded; the lab's genomes live in liulab-genome, so ask it for a path. It is the one section here with no committed output behind it, because a genome cannot be committed.

How to read the sheet¶

Open primers.tsv in a spreadsheet or an editor. One row per oligo: the name you order it under, the bases 5' to 3', how many there are, and the melting temperature. Both oligos here are 18 bases, at 61.7 and 65.3 °C.

That temperature is the binding part's, not the whole oligo's. The annealing temperature on the summary line comes off the lower of the two, by the polymerase maker's own rule.

The sheet carries no verdicts; the summary line's last words hold the worst of every check. Two checks are never judged: the melting temperature of the whole oligo, and how tightly its last bases hold. Nothing sourced says what a good value is.

Before you order¶

• Read the summary line: both sequences, the amplicon, the annealing temperature.
• Check the amplicon is the region you meant, both ends counted in.
• Check each tail reads the way the enzyme needs it.
• Set the block to that annealing temperature.
• On genomic DNA, read genome.tsv: the only amplicon should be yours.

Tips and troubleshooting¶

The pair warns and you want a cleaner one. More room is the only thing that helps. Move the ends of --region a few bases, or aim at another temperature with --target-tm.

Your primer needs a tail. --forward-tail and --reverse-tail join bases to a 5' end. The tail stays out of the search for a binding site, and it still lengthens the amplicon.

No index sits beside the genome FASTA. The region is read through that index, so the run stops without it. Ask liulab-genome for an indexed copy.

Where the numbers come from¶

Every check is held to a band with a source: primer3's defaults for the hairpin and dimer limits, IDT's ranges for length and melting temperature, NEB's calculator for the gap between a pair's two temperatures. Where no source states a band, the package either proposes one and says so, or judges nothing.

Melting temperature is the nearest-neighbour model, computed with primer3 and salt-corrected for the polymerase's own buffer, so it agrees with NEB's Tm Calculator. The annealing rule and the extension rate are that polymerase's, and the off-target rules are Primer-BLAST's defaults.

pixi run mbio primers design --help lists the rest.

References¶

• SantaLucia, J. (1998) A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. PNAS 95, 1460–1465.
• Ye, J. et al. (2012) Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics 13: 134.
• NEB, Tm Calculator, and the Q5 High-Fidelity DNA Polymerase protocol (NEB #M0491).