cloning inabundant 3' ends

Keith.A.Johnson at DARTMOUTH.EDU Keith.A.Johnson at DARTMOUTH.EDU
Thu Mar 19 21:28:03 EST 1992


I have been attempting to clone the 3' end of an inabundant transcript to
complete the sequencing of the gene as part of my thesis work.  I have cloned
most of the gene from cDNA libraries generated with either oligo-dT or random
hexamers and oligo-dT, but have not been able to clone the 3' end of the
gene.  To date I have screened more than 10 cDNA libraries.  I have also
tried to reverse transcribe polyA+ RNA and then to use the RT-product in a
PCR amplification utilizing the primer/adaptor from the RT reaction with a
specific oligo for my gene which resulted in a brief glimmer of hope which
ended up being a false positive.

Is there anyone out there who might be able to suggest an experiment to help
me clone the 3' end of my gene or who has had similar difficulty in cloning
the 3' end of a gene?  Are there any published accounts of transcripts whose
3' untranslated region was unstable when found in a plasmid?

Thanks in advance for any assistance.

Keith A Johnson
Dartmouth Medical School
Hanover, NH  USA



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