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P-element insertion question

Robert M. Mihalek mihalek at FORMULA1.smtp.anes.upmc.edu
Thu Feb 4 16:49:13 EST 1999


In article <o0ku2.1183$t5.8452712 at WReNphoon2>
jbanders at helix.nih.gov (John Anderson) writes:

> I have a simple question regarding the behavior of P-
> elements:
> 
> I know that when a P-element mobilizes, it can excise
> imprecisely, taking away some flanking DNA. My question
> is, when this occurs and then the P-element reinserts
> somewhere else in the genome, does it insert precisely,
> losing the flanking DNA or can it insert the flanking
> DNA into the new site?
> 
> I want to know because I'm trying to create a deletion
> by mobilizing a P-element that is near my gene. I want
> to use a PCR screen, with one primer in the P and one
> outside it. A positive would indicate that the P is
> still where it was. It has occurred to me that I could
> get false positives, if the P-element and the flanking
> DNA where the 2nd primer is are still together, even if
> they have moved.
> 
> Please respond by e-mail or to the newsgroup. Thanks in
> advance.
> 
> John Anderson
> NHLBI

I am almost positive that the flanking DNA would get lost during the
re-insertion process, since re-insertion requires the specific
sequences on the end of the P-element to be recognized by the
transposase. However, you may want to get around the problem all
together by just screening excision events via Southern blot, looking
for a change in restriction fragment length upon excision. Or, design
your primers so that you don't amplify anything with P-element
sequences. With some of the PCR kits available, you can routinely
amplify >7kb regions,so you'd pick up even pretty big deletions. (I'm
assuming here that the P-element doesn't carry any markers, because
your first line of defense would be to screen against the marker
genetically, but you could get burned by internal deletions taking out
just the marker sequences of the P-element.)



Bob Mihalek=================================================
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