Site-directed Mutagenesis Oligos

Karl Fischer kfischer at gpu.srv.ualberta.ca
Mon Mar 20 16:21:08 EST 1995


In article <DEZWAAN_T-2003952035250001 at roof-physiol.med.upenn.edu>,
DEZWAAN_T at a1.mscf.upenn.edu (Todd DeZwaan) wrote:

> I would like to design a mutagenic oligonucleotide that carries four
> mismatches spread over 17 nucleotides.  There is quite a bit of base
> pairing that occurs between the mismatches.  My question is how do I
> design the oligo to account for the stability conferred by this internal
> base pairing?  If I just disregard this base pairing and tack on 12 - 15
> perfectly matched nucleotides on either side of the 5'-most and 3'-most
> mismatches (as suggested by Sambrook, et al.) I come up with a 50-mer. 
> This seems excessive and potentially problematic with regard to secondary
> structures and primer dimers.  I would greatly appreciate any help on this
> problem.

Todd,

With four mismatches over 17 nts, you can probably get away with 8-9 nt
long flanking sequences to your 5' and 3' mismatches...my suggestion is
run your minimal mutagenic oligo through a program like Amplify and check
the primability and stability percentages BEFORE you send your 50 mer for
synthesis; shift the oligo or add nts one at a time to the ends if false
priming is predicted to occur. I normally use this approach and it
minimizes the size (and extra expense) of the mutagenic oligo I ultimately
use.

Cheers

Karl

-- 
Karl Fischer
kfischer at gpu.srv.ualberta.ca
tyr-2 at bones.biochem.ualberta.ca



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