primer design problem (View forum version)


Posted 16 December 2009 - 08:49 AM

i am designing primer for SNP-RFLP and no restriction enzyme is available to cut the SNP site.
so, i make an artifical restriction site by inducing a primer mismatch in the primer, so that the pcr product can be cut by enzyme.

however, the mismatch i induced is near the SNP site, and after cutting by enzyme, the fragment is too short that cannot be visualized...
someone said i can add a poly(T) tail to the 5' end of the mismatch primer so that the fragment can be long enough.

anyone know whether it will work???


Posted 16 December 2009 - 09:15 AM

Yes, this will likely work. There is nothing magic about poly T -- an arbitrary sequence would also be fine (one that doesn't also get cut by your enzyme, of course).