strange cloning results

Frank O. Fackelmayer fof1 at chclu.chemie.uni-konstanz.de
Mon Jun 15 09:48:01 EST 1998


William Sun wrote:
> 
> Hello all,
> 
> I've isolated several clones from a cDNA library with a labeled probe.  When
> I analyzed the clones, I found that many of them are in the reversed
> orientation (the library was oligo-dT primed and uni-directionally cloned).
> Is this just an artifact or can some gene have 2 ORFs in opposite directions?


just a common cloning artifact. In library construction, there are inevitably
events that might lead to junk clones, like reverse orientation, or the poly-A
artifact you describe below, or head to head ligated cDNAs (usually those very
long clones you first believe to be full-length, sigh...). There´s no other
way than to accept this, and simply use the other clones you have found. For
comparison: I had some 10-20% of clones in reverse orientation, and below 5%
head to head ligated cDNAs. The poly-A artifact is possibly in the same range,
possibly even lower, but I had to few to do statistics.


> Another strange clone that I isolated was that if I translate the aa in the
> same frame as an already known homologous protein, I don't detect a stop codon -
> it translates right through to the poly-A tail.  I sequeced the clone in both
> directions so I'm sure it's not a mistake in the sequencing.  Can anyone offer
> an explanation?

yeah, its plain simple. Your cDNA most likely has a stretch of A´s where the
oligo-dT primer annealed during reverse transcription. So, the poly-A end you
see is not the poly-a tail of the mRNA but the one "encoded" by the oligo
dT-primer. In fact, A-stretches as short as 5-6bases might lead to this
artifact in a low percentage of clones. These clones usually end with a poly-A
tract where the cDNA had the A´s. Consequently, they often lack a stop codon
before the poly-A tract.

Hope this helps,
Frank



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