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
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.
More information about the Methods