Inosine in PCR primers

[Peter J. Neame]

Chrystala Constantinidou (C.Constantinidou at bham.ac.uk) wrote:

: Does anyone have experience of using inosine in a PCR primer. I have read
: that it is not a good idea to have them too close to the 3' end - has
: anyone used inosine and how close is too close. What is it about inosine
: that may make it unsuitable to be directly at the 3' end?

The dogma has it that you have to have a precise match at the 3' end of a 
primer. However, a bit of "slop" is actually tolerated. It depends a lot 
on the rest of the primer (probably). Regardless, the worse the match at 
the 3' end, the more low specificity you are likely. We've had as many as 
4 inosines in a 20-mer, but never have them in the last or penultimate 
position. 

haven't done a rigorous study, however.

Good luck

Peter Neame                                                              
Dept Biochem. and Mol. Biol., USF, Tampa,                                
Florida.