Primer3 in Biopython

Question

[calyssa7]

I am new to Biopython and I would like to know how to automate primer design (I have about 100 sequences that I would like to automatically design primers to). Could someone please provide detailed instructions for how to use Biopython to do this?

Answer 1

[piet]

You basically want to batch run primer3 to design more than 100 pairs of primers. You do not have to use Biopython for this purpose, pure python or a shell script should be enough.

In any case you will call the standalone primer3 program as a subprocess, passing it an appropriate input file and reading the results from its stdout. Primer3 input and output is a simple stream of key-value pairs, one pair per line. Please make yourself familiar with this format. Then write an input file for a single target sequence, run it through primer3, and fiddle out the best primer pair from the output. If you have managed that for one target, it will be easy to write a script to run a batch of 100 targets.

[John]

Piet's totally right, but as a word of warning do check the primers you get out of primer3 manually. It's pretty good, but occasionally it gets it totally wrong. In particular, check that the primers are unique to the genome with BLAT, double check the Tm deltas for the two primers, and ideally check for a hairpin loop in the amplicon with UNAFOLD that would prevent the primers from annealing. Or use that automated thing from Ensembl which probably does all of that for you. I forget what it's called though :(