Site-directed Mutagenesis Oligos

Emmanuel Skoufos, Ph.D. skoufos at nefeli.imbb.forth.gr
Tue Mar 21 13:25:12 EST 1995


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

Which method are you planning on using for generating the mutations?
You can use inverted PCR and come up with smaller oligos (about 30mers
should work).

Emmanuel





_______________________________________________________________________________

Emmanuel Skoufos, Ph.D.
Insect Molecular Biology Group
Institute of Molecular Biology and Biotechnology
Box 1527 
Heraklion, Crete 71110
Greece



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