random Mutagenesis

[Paul N Hengen]

In article <3um9ef$6on at bubba.NMSU.Edu> dkim at nmsu.edu (Daniel Kim) writes:

: I need to do random mutagenesis on a 3kb fragment.
: Is there any efficient way of doing the mutation,
: say by PCR?

| There is a method called DNA shuffling mutagenesis by a guy named Willem 
| Stemmer, published in SCIENCE and/or PNAS in late 1994/early 1995.  He 
| takes a PCR fragment, DNAse I digests it and purifies out 50 - 200 bp 
| fragments, then run PCR cycles with NO primers to let the fragments 
| anneal and primer off each other, introducing recombination errors as 
| well as PCR misincorporation errors.

@article{Stemmer1994a,
author = "W. P. Stemmer",
title = "Rapid evolution of a protein {{\em in vitro}}
by {DNA} shuffling",
journal = "Nature",
volume = "370",
pages = "389-391",
comment = "DNA shuffling mutagenesis",
year = "1994"}

@article{Stemmer1994b,
author = "W. P. Stemmer",
title = "{DNA} shuffling by random fragmentation and reassembly:
{{\em in vitro}} recombination for molecular evolution",
journal = "Proc. Natl. Acad. Sci. USA",
volume = "91",
pages = "10747-10751",
comment = "DNA shuffling mutagenesis",
year = "1994"}

I would be interested to hear the experiences of anyone who
has tried this procedure.

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