primer specificity/mismatches

Simone Marker via (by marker from
Thu Apr 5 04:14:16 EST 2007


does anyone have experience with primer design for specific RT reactions or 
in general?
I want to find primers for a gene from a multigene family, in which the 
family members are highly similar in some regions. I'm not sure about the 
creteria for specificity: how many mismatches are necessary? For RT-primers 
I normally avoid primers that have only mismatches in the 5' moiety. But how 
many mismatches are necessay at the 3' moiety of the primer to guarantee 
specificity? I tried to chose at least 3 mismatches. Is a primer ok that has 
only one or two mismatches at the last 3'base(s) and no mismatch at the 
residual bases of the primer?

Do I have to consider any differences between RT and PCR in general or do I 
have to apply the same criteria for primer specificity? I use an RT enzyme 
that is able to reverse transcribe at 50-60°C, so that I can adapt my 
primers very flexible (normally I use primers with a melting temperature of 
55°C and do the RT at 57°C, works well).

Thank you very mutch in advance,

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