I'm working on a script using BioPerl's implementation of Primer3 in the Bio::Tools::Run::Primer3Redux package.
This question may be just a result of paranoia - but when you specify a region to be included for consideration for primer design (using the SEQUENCE_INCLUDED_REGION attribute) - does Primer3 still include the rest of the input sequence to check for possibility of mis-priming or off-target effects?
I would assume yes - but it's not 100% clear from the documentation.
Those numerous parameters are described in the Primer3 manual and that may be the documentation you are referring to. In any case, here is what the Primer3 manual says about SEQUENCE_INCLUDED_REGION:
A sub-region of the given sequence in which to pick primers. For example, often the first dozen or so bases of a sequence are vector, and should be excluded from consideration. The value for this parameter has the form
[start], [length]
where [start] is the index of the first base to consider, and [length] is the number of subsequent bases in the primer-picking region.
It sounds to me like it will not consider the rest of the sequence by default, only [length] bases after [start]. So, my interpretation would be that Primer3 is not checking anything beyond that range.
Hrmm.. I know it won't consider those regions as valid primers - but I'm wondering if it includes regions outside the "included area" for mispriming purposes. I'll have to test it out to see what the real functionality is by giving it contrived sequence.
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