cDNA from procaryotes - how ?

Laurel Lenz vendredi at u.washington.edu
Wed Aug 13 21:03:10 EST 1997


In article <zakaria-1308971141230001 at 153.96.224.92>,
Hayssam Zakaria <zakaria at ita.fhg.de> wrote:
>I intend to create a cDNA-library out of a collection of microorganisms to
>screen for new genes. While the creation of cDNA's from eucaryotes is
>pretty easy the lack of polyadenylation (poly-A) is a problem in
>procaryotes.
>So does anybody have an idea how to get a cDNA from procaryotes esp. with
>full length clones or how to establish an expression library.
>Any idea or reference would be of big help.
>
>   Hayssam Zakaria
>

Since procaryotes lack introns, you can clone fragments of bacterial genomic
DNA into a vector of choice and possibly get expression in your host bacterium.
You may want to clone large fragments - and thus hopefully entire operons - if
you hope to get expression under endogenous (donor) promotors.  Alternatively,
you could try cloning smaller fragments behind a promotor of your choice. Of
course these approaches both have caveats and neither would probably give you
a complete representation of expressed genes from the donor bacterium.  However,
we have successfully used the 1st approach to clone a Listeria gene (if you're
interested, see "Immunity 5:63 (1997).")

Good luck,
Laurel




More information about the Microbio mailing list