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2-hybrid libraries

2-hybrid libraries - Protocols and Methods Forum

2-hybrid libraries - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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Old 09-01-2005, 02:35 AM
Chris Fisher
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Default 2-hybrid libraries



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. 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.

Thanks, Chris

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Old 09-01-2005, 07:33 PM
Peter Ellis
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Default 2-hybrid libraries

[Only registered users see links. ] wrote:

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.


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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