gene walking techniques

Andrew Hobbs andrewh at uniwa.uwa.edu.au
Sun Jul 4 21:03:53 EST 1993


APPLING at UTBC01.CM.UTEXAS.EDU wrote:
: We have been trying for several years now to isolate genomic clones containing
: the 5' flanking region (promoter) of a rat gene.  After searching through
: 3 different libraries with numerous probes and PCR techniques, we have never
: found any authentic sequences upstream of the initiator codon (although we
: have come across a psuedogene a few times).  We have apparently found the 5'
: end of the mRNA using reverse transcription-PCR, but using this as a probe has
: not yielded the corresponding genomic clone.  We are forced to conclude that
: this region does not exist in conventional genomic libraries.

Hi,
One suggestion that may explain the problem.  Have you
thought of the possibility of a  (very large) intron very close to the
transcription initiation site.  This has caused problems in finding the
start of a number of genes since the small 5' exon isn't long enough
to give a decent signal using regular labelled probes.  How much of the 5' end
fragment prepared using PCR do you have and how was it labelled.  It may be
worthwhile getting an oligonucleotide synthesised and using that to
screen your genomic library.  How long is the 5'non-coding region.  Have
you tried gene walking?  Have you tried doing a Southern using just the
5'end of the mRNA; the bit not represented in your current genomic
clones.  If you can't get a hybridization signal then you'll never find
the corresponding clone.

Hope this helps

Andrew Hobbs
Biochemistry
University of Western Australia.
andrewh at uniwa.uwa.edu.au 



: We are thus ready to try a PCR-gene walking technique and are looking for
: advice from experienced users of the various strategies such as inverse PCR,
: single-site PCR (PCR Methods & Applications 1:129-135, 1991) or panhandle PCR
: (Nucl. Acids Res. 20:595-600, 1992).  Any comments on these or other techniques
: will be appreciated.

: Thanks
: _______________________________________________________________________________
: Dean R. Appling
: Dept. of Chemistry & Biochemistry
: Univ. of Texas at Austin
: Austin, TX 78712
: (512)471-5842
: (512)471-5849 (FAX)
: appling at utbc01.cm.utexas.edu
: _______________________________________________________________________________



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