Site-directed Mutagenesis Oligos

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

In article <DEZWAAN_T-2003952035250001 at>, DEZWAAN_T at (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
>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 Skoufos, Ph.D.
Insect Molecular Biology Group
Institute of Molecular Biology and Biotechnology
Box 1527 
Heraklion, Crete 71110

More information about the Methods mailing list