5' Primer modification - Tm-effect ?? (Dec/13/2006 )
does anybody know the effect of a 5' modification (e.g. adding biotin) on the Tm of a primer?
I would assume that the Tm becomes lower, because the primer perhaps cannot bind completely to template (i.e. steric problems). But I'm not sure, if this happens and if it is important as 5' is not so important if it binds or not.
Has anybody experience?? To try out myself is quite expensive
Many biotin modifications include a substantial linker to avoid steric problems. The important part of binding a primer is the binding of the 3' end (recall that you routinely add 5' bases which don't match for restriction sites etc.). I think you have every reason to believe that a 5' biotin modification will function very nearly the same as an unmodified oligo.