Disappearing Clones during Library Screening

Jeff Dean jdeanx1 at UGA.CC.UGA.EDU
Tue Sep 6 14:03:06 EST 1994


Dear Molecular Bionauts:
        
        Here's a mystery to start the fall semester.

        In a recent report (1994 BioTechniques 16:98-103), Amaravadi & King
describe difficulties they had in isolating a cDNA for c-mycb from a Xenopus 
laevis library. Using PCR, the library gave a definite postive signal in
initial screening runs, but as the library was diluted the signal eventually
disappeared. These researchers eventually settled on a hybrid protocol in 
which library dilution was monitored with PCR for the first couple of 
screens, and then they switched to plating and plaque coring.
        We have recently seen a similar phenomenon in our attempts to clone a
cDNA encoding a particular cell wall enzyme from yellow-poplar. The vector is Stratagene's UniZAP and the host is XL-1 Blue. The oligo probes do a fine job 
in PCR screening for the first couple of rounds, and then the signal 
disappears. It would appear that clones of this cDNA are not getting 
amplified when phage are replicated after dilution. We don't think that leaky 
expression of the gene product is a problem since ALL positives we have 
pursued disappeared (only one in three should do that if expression from our 
unidirectional vector is 'lethal'). The full-length of the cDNA insert is 
around 2 kB, so we don't think that insert size is leading to slow 
replication or poor assembly. It could be a DNA sequence-specific phenomenon,
but we haven't found any obvious motifs in PCR fragments cloned directly into
TA vector.
        All this leads one to wonder how often this problem occurs, and how 
many Ph.D. candidates have foundered on the problem. It sounds reminiscient
of problems previously cast in the guise of 'rare' message quirks. I suspect
that this phenomenon is more common than sometimes thought, and that we are 
going to hear much more about it now that PCR-based library screening is 
becoming so routine.
        So... What causes poor phage replication with certain inserts? Or, is
some other phenomenon at work here?

I'm looking forward to reading your thoughts on this! :-)

-Jeff
***********************************************************************
*                              *                                      *
* Jeffrey F.D. Dean, Ph.D.     * "Ay," quoth my uncle Gloster,        *
* Department of Biochemistry   * "Small herbs have grace, great weeds *
*    and Molecular Biology     *    do grow apace:"                   *
* University of Georgia        * And since methinks, I would not grow *
* Athens, GA  30602-7229  USA  *    so fast,                          *
*                              * Because sweet flowers are slow, and  *
* jdeanx1 at uga.cc.uga.edu       *    weeds make haste.                 *
* (706) 542-1710               *                                      *
* (706) 542-2222 FAX           *      - Richard III, Act II, Sc 4     *
*                              *                                      *
***********************************************************************




More information about the Methods mailing list