Site-directed Mutagenesis Oligos

Todd DeZwaan DEZWAAN_T at a1.mscf.upenn.edu
Mon Mar 20 21:35:25 EST 1995


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 DeZwaan



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