The default settings for Primer3 do not actually check for whether a primer will bind at multiple places in the template. To turn this on use the PRIMER vs PRODUCT setting in the PRIMER BINDING tab (the Primer3 parameter is “PRIMER_MAX_TEMPLATE_MISPRIMING”). The default value is -1, which means do not check. Change this to 1 to start checking, or increase the number to increase stringency.
This should now find any duplicate alignments.
Incidentally an upcoming release will have a special “QuickPrimer” tool for checking such issues as well as the Primer3 tool.
