2-hybrid libraries

Peter Ellis pjie2 at cam.ac.uk
Thu Sep 1 14:33:40 EST 2005


cfisher1 at chartermi.net wrote:
>I've isolated an interesting cDNA from a human Clontech library that could
>only have been made via transplicing from mRNAs originating from 2 different
>chromosomes.  It seems to be real, but before I follow up on it I was
>wondering if anyone with experience in cloning has found cases where similar
>artifacts might have arisen in library construction, or of other possible
>problems with commercially available libraries. Or perhaps have advice on
>how I might tell if it's an artefact of library construction.  

Chimeric clones are far from uncommon. I've seen this many times 
before, especially when making subtracted libraries.  The PCR-Select 
cDNA subtraction kit from Clontech involves a restriction digest of the 
double-stranded cDNA, followed by a blunt end ligation step to add on 
the adaptors. At this point it's easy to ligate two separate inserts 
together.

If it's a subtracted cDNA library, this is most likely the cause.  If 
so, it's quite likely (but not 100%) that there will be an Rsa1 
restriction site (GT'AC) at the join between the two.

>
>In this case
>the putative exon boundaries don't seem to follow what's been published for
>the genes in question, but with splice donor/acceptor sites being pretty
>degenerate in humans I don't really know if this is a big red flag or
>not--the whole cDNA encodes a rather interesting open reading frame.

The easy way to confirm if it's real or not is to design an RT-PCR to 
amplify across the join, and see if you can recover a clone from the 
same tissue that was used to make the library.  If not, it's almost 
certainly a library construction artefact.

Even if you do confirm that there's a genuine transcript matching your 
clone, I'd be wary of claiming trans-splicing - there's more than you 
think lurking in the "heterochromatic" holes in the genome sequence, 
and jumbled/recombinant loci are among them.

Peter Ellis


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