gene walking techniques

Tue Jun 29 08:24:23 EST 1993

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.

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.

Dean R. Appling
Dept. of Chemistry & Biochemistry
Univ. of Texas at Austin
Austin, TX 78712
(512)471-5849 (FAX)
appling at

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