thanks for your reply
would you please sent me the software link again?
yes. I want to do sequencing after the PCR.but the problem is the high similarity (97%) of the psuedogenes sequence with the original gene.and I have no choice except designing primers at the regions with 1 or 2 mismatches from the psedogenes...
so I 'm not sure if the psuedogenes would amplify to some extent with the original fragment or not at the end
i noticed that the orientation of the original gene at GeneCards browser is:
minus strand (Start: 2,138,711 bp from pter End: 2,185,899 bp from pter )
and some psuedogenes are in:
plus strand (Start: 15,029,565 bp from pter End: up to 16,471,364 bp from pter)
does it mean that my forward and reverse primers from the original gene cant amplify these psuedogenes?