Hi Misbah Hussain

You can optimize annealing temperatures for the primer actually in the lab at range of temperature exp: (from 50 up to 78)C and will show you which temperature is really appropriate depending on strongest electrophoresis bands. According to my experience does not show the same temperature as estimated by the program.

I would try doing the pcr in the presence of 1Molar betaine ( very cheap from many suppliers) which lessens the effect of the triple hydrogen bond in high GC amplimers and makes them melt easier. You could also design DCAPS primers which have a deliberate second mismatch so create a restriction enzyme site when each of the alleles is present. This always gives a positive result...amplimer then digest so may be preferable to ARMS but may still need to be amplified in presence of betaine. see

http://helix.wustl.edu/dcaps/dcaps.html  to design primers

Yes, you can try with these primers. Use 5% DMSO in your PCR. We have  optimized assays for few SNPs between 62-68 degree C annealing temperature which included primers showing high Tm.

Thank you so much everyone.

I am surly gonna try these. I hope they will work.   

Hi Misbah Hussain

You can optimize annealing temperatures for the primer actually in the lab at range of temperature exp: (from 50 up to 78)C and will show you which temperature is really appropriate depending on strongest electrophoresis bands. According to my experience does not show the same temperature as estimated by the program.

Hi Shaden M H Mubarak ,

Thanks for your answer. Yeh I experienced the same in my experiments.

I tried the mentioned primers and they worked very well at 69 degree.