library screening

Dom Spinella dspinella at chugaibio.com
Thu Jun 26 10:50:01 EST 1997


Deborah Britt writes:

> We recently screened a Stratagene Reuber hepatoma (rat) library, and
> pulled out six clones. All six turned out to be cytochrome C oxidase. 
> Needless to say, this was pretty disappointing, after all the work that
> went into it. We don't have a specific DNA probe, and were using cDNA
> probes from various cell lines that do and do not express the protein we
> are trying to clone, and I think somewhere at the beginning we managed to
> select/amplify the cytochrome C clones.
> 
> I am just curious now about library representation or biases. Does anyone
> know of a reference, or a web site, where I can get information about
> sequence representation in expression libraries? Also, if anyone has had
> experience (good or bad) screening this particular library, I would be
> happy to hear about it.
> 
> Thanks!

Deborah:

Well, you don't say which subunit of cytochrome c oxidase you pulled
out.  Some of the subunits are encoded by mitochondrial genes, and we
have observed that differential cloning methods such as subtractive
cloning, differential display, etc. often pull out mitochondrial genes
(especially cyt-c oxidase) -- presumably due to differences in the
numbers of mitochondria in various tissues or samples.  Cytochrome c
oxidase transcripts (both nuclear and mitochondrial-encoded subunits)
are very highly expressed in most tissues.  The entire mitochondrial
genome is only 16.5 kb and perhaps you can use it as a hybridization
probe in filter lifts to eliminate any Mt genes from further
consideration.  For an overview of cDNA library representations, you
might check the big EST databases to see which genes are highly
represented in many libraries.  A large review was published in late
1995 by HGS in the Genome Directory Supplement to Nature and can provide
you with a great deal of insight.  See Adams et al. Supplement to Nature
377: 3-175 Issue no. 6547S (September, 1995).  Good Luck.  -- D.G.
Spinella



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