dkim at NMSU.Edu
Wed Mar 18 14:55:14 EST 1998
You might want to look at W. Stemmer's "DNA Shuffling Mutagenesis"
technique. I do not remember specifically where I read it, but a search
for Stemmer or DNA Shuffling should be fruitful. Briefly, your target DNA
is lightly nicked with DNAse I . The fragments are then recombined using
PCR with no primers. After many cycles, the original fragment is
regenerated, and can be amplified by primer-driven PCR from the partial
fragments. The full-length product is then cloned.
This method can also be used to combine the sequences of two or more
different, but related, genes for "in vitro evolution".
dkim at nmsu.edu
Vladimir Svetlov <svetlov at oncology.wisc.edu> wrote:
: In article <bbeitzel-1603980809000001 at idl04.salk.edu>,
: bbeitzel at biomail.ucsd.edu wrote:
: > Hola,
: > Just wondering if anyone had any suggestions about mutagenesis. I have a
: > cDNA that I would like to mutagenize to give me a pool of different
: > mutants (point mutants and small deletions.) Any recommendations as to
: > the best way to go about doing this?
: No random mutagenesis protocol will reliably generate small deletions,
: point mutations can be easily generated by Taq-driven PCR in presence of
: Mn++ and/or high dNTP concentrations. Use a lot of template and a few
: cycles (20) to avoid propagation of a few mutations "early" in
: amplification throughout the population of PCR products. More laborious
: techniques include addition of an excess of one of the dNTPs or dITP, but
: these also have some bias in the kind of mutations you are getting.
: Vladimir Svetlov
: McArdle Lab for Cancer Research
: Dept. Oncology
: 1400 University Ave.
: Madison, WI 53706
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