Stephen J. Palmieri (rocco at mail.utexas.edu) wrote:
: I am interested in randomly mutagenizing a particular gene of choice and
: was wondering if anyone out there knows of a reliable protocol that can be
: modified to generate a single point mutation per DNA molecule. Thanx in
: advance.
I'm not certain if this will work for your problem; but someone I used to
work for, Steve Nordeen, developed a method a few years ago for
introducing random mutations into a short stretch of DNA. It involved
standard oligonucleotide synthesis, except each of the four pools of
phosphoramidites was doped with a small amount of the other three
nucleotides. Thus when a strand was synthesized there was an occasional
random "misincorporation" of a base thus generating a mutant strand. This
strategy probably works best for short stretches of DNA, but might be
applicable for targeting a particular region of your gene of interest,
otherwise, I'd probably go with the PCR techniques described. My guess is
that the PCR misincorporation techniques might work best if you did the
reaction using "nonoptimal" conditions, such as limiting one or more
nucleotide concentrations or perhaps Mg++ or salt concentrations.
Good Luck
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L. Scot Bastian Ph.D. 206-762-1010 ext. 1918 (voice)
University of Washington at the 206-764-2689 (FAX)
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