Finding Target Genes for DNA Binding Proteins

Paul N Hengen pnh at ncifcrf.gov
Fri Oct 4 11:15:58 EST 1996


William J. Meilandt (wmeiland at lonestar.jpl.utsa.edu) wrote:

> We have a putative DNA binding protein and would like to identify the 
> genomic sequences to which the potein binds to.  Does any one have 
> references or ideas they would like to share?

Have a look at this paper, then point your web browser at
http://www-lmmb.ncifcrf.gov/~toms/sequencelogo.html for more
information regarding the method described.

@article{Schneider.Stephens.Logo,
author = "T. D. Schneider
 and R. M. Stephens",
title = "Sequence Logos: A New Way to Display Consensus Sequences",
journal = "Nucl. Acids Res.",
volume = "18",
pages = "6097-6100",
year = "1990"}

> I am considering to purify the protein and put it on a column and then 
> pass predigested genomic DNA thru the column to isolate genomic fragments 
> which bind to my protein.  Is it better to used protein purified from my 
> tissue of interest or in vitro translated?

Definitely in vitro translated or better yet expressed in E. coli if you can
since you'll get better yield and it should be relatively free of other
contaminating DNA binding proteins.  Also consider gel shifting randomly
generated DNA and then isolating the shifted material for cloning and
sequencing.

> An alternative approach would be to screen a genomic library with the 
> putative binding site for the DNA-binding protein. Isolate this clone, 
> digest it into smaller pieces, clone into a vector and re-screen with 
> the binding site, and sequence the positive clones. 

Better done after you have a handle on the binding properties of
the protein and the binding site sequences to which it binds.

--
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