I have a 2kb region of DNA, and I need to design five non-overlapping primer pairs to any location within this same stretch of DNA. I need to use all of these primers together in the same PCR reaction, so they must be multiplex compatible (e.g. not have any complementarity to each other). Does anyone know of any software that can do this automatically? Or of rules for filtering a whole load of Primer3 output primers to select the top five that might work in a multiplex pcr?
Thanks for the help!