cDNAs & ORFs

Andrew Hobbs andrewh at uniwa.uwa.edu.au
Fri Jun 4 02:22:48 EST 1993


Mark Bradley (MPB at CBR.DWE.CSIRO.AU) wrote:
: Dear Netters:
: A query: we have pulled a cDNA from testis which contains an ORF for
: a sperm antigen.  However we have found a curious(?) poly-T tract 
: inframe with the start of the first ATG.  The poly-T tract isapprox
: a 30mer.  
: Question: does anyone know the significance of the seq prior to the 
: first start codon and how common(?) is such a seq in the apparently
: non coding region adjacent to a complete ORF.
: Any comments would be most welcome

: Mark Bradley
: CSIRO
: Div Wildlife & Ecology
: Canberra
: Australia
: Email: mpb at cbr.dwe.csiro.au

Hi, 

I haven't ever seen an occurence in a published paper.  However I
have obtained an aberrant run of T's (or A's) due to the oligo dT
priming off a short poly A stretch during cloning.

Therefore my first question would be "have you confirmed that it is
real and not a cloning artifact?": ie is it in more than one
independent clone.  Could it have been derived from oligo dT used
during the construction of the cDNA library; ie by unwanted priming off
a shorter sequence with a high proportion of A's in the cDNA sequence. 
Also, is it embedded in sequence or at the end of the cloned insert? 
If the latter, another possibility could be that it was ligated in tandem
with the real insert during library construction (ie as an dA.dT double
stranded fragment derived from fragments of poly A tail).

Hope this might help.

Andrew Hobbs
Dept of Biochemistry
University of Western Australia

andrewh at uniwa.uwa.edu.au



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