Size in bp of wild type band for 1st set of primers e.g. 4000

Range that wild type band can vary

Allow primers to be chosen in neighbouring genes*

Discard primers which give multiple bands (takes alot longer to calculate)

What primer size is preferred (note that to attain desired annealing consistency actual size are allowed to vary)

Max distance from 1st primers that the second set can be e.g. 200