RTPCR/degenerate primers with inosine?

Cynthia Fisher clfisher at oci.utoronto.ca
Tue Oct 18 12:12:18 EST 1994

I'm designing degenerate primers to use in RT-PCR reactions
and I'd like some advice regarding the use of inosine bases.

My primers will be 16-20mers, and to keep the degeneracy down
to a moderate level (about 216-fold) I need to use 6 inosines in
my forward primer, and 3 inosines in my reverse primer. 

Are there any 'rules' about the most effective placement
of the inosines e.g. can they be adjacent to one another,
or will this negatively affect annealing? 

Also, what is the minimum acceptable Tm for degenerate primers?
I'm looking at a min Tm of 38 degrees C for one of my primers -
has anyone any opinions on whether this will work (provided my
first few annealing cycles are carried out at a lower temperature
like 35 degrees C).

Please respond by email directly to me (and post to the group
as well). Thank you all!

Cynthia Fisher
clfisher at oci.utoronto.ca

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