RTPCR/degenerate primers with inosine?

Robert.Coelen at jcu.edu.au Robert.Coelen at jcu.edu.au
Wed Oct 19 18:25:36 EST 1994


In article <Pine.SUN.3.90.941018130300.29815B-100000 at cyto> Cynthia Fisher <clfisher at oci.utoronto.ca> writes:
>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? 


You haven'tgot a whole lot of space to move if you want to use 6
inosines in a 16-mer. So try to use longer oligos if you can. I would
avoid inosines in the last three (3') positions. As to bunching them up, 
I suspect that such a configuration depresses the Tm more than having some
fixed bases in between. On the whole, spend your degenerate positions on the
3' end and use the inosines towards the 5' end though.

I have heard of primers that work even at 30 dgc ! and I wouldn't 
necessarily go to 35 dgc for your primers with a SUPPOSED Tm of
38, start by using 40 dgc and work back.


Hope it helps, but......... no guarantees !


Cheers,



Robert

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Dr Robert J. Coelen                                  fax:   61-77-791 526
Dept of Biomedical and Tropical Veterinary Sciences  phone: 61-77-815 024
James Cook University of North Queensland
Townsville-just-about-on-THE-reef, Q, 4811
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