The search of an automated Primer-Pair Search Tool from Genomic Coordinates

I have a problem: I need to design 96 different primer pairs to produce 96 different amplicons from extracted mouse genome. Each amplicon should derive from a specific genomic region, and avoid off-targets.


A list of 96 mouse genomic regions (mm10, format: Ch4:18,405,405 – 18,405,805) that I would like to make an amplicon from.


A table with a Fw and Rv primer pairs that will amplify that region. The primer pair should:

  • pass the Primer3 test (control for Tm and CG content and simple things like that)
  • pass a BLAST test (control that the primer pair is unlikely to give rise to a secondary amplicons from an off-target region)

This tool already exist: Primer-BLAST available at NCBI. But that system doesn’t allow me to input multiple FASTA files at once. That means that I can spend a day clicking on my browser to get the work done.

I am very confident this problem was solved by people smarter than me, but for the sake of me I haven’t found anything online. I have seen scientists suggesting to use Primer-BLAST from a command line, but I didn’t find instructions for it.

HELP! I am decent at Python, R and API.

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