P-element mutagenesis and flanking DNA.
We are currently doing a similar experiment except we want to insert and
not remove a P-element in our gene. We also screen by PCR with one primer
facing out from the P-element and a battery of primers specific for our
gene. One of our 'gene-specific' primers is, unfortuately, not so specific
since it gives PCR products at several different P-insertion sites. We have
sequenced several of these insertions and found that the sequence outside
the P-element is always different also in a set of 4 insertions in the same
gene. If these were false positives due to moving of flankíng DNA then the
DNA next to the element should be the same in these 4 cases.
We would be very interested to hear from people with experience in PCR
based screening of P-element mutagenesis. What is the optimal number of
primers that can be used simultaneously? What is the optimal distance
between gene specific primers? What is the best stage to use (we use
embryos)? Are there some genes that cannot be mutated or insertions that
cannot be found?
Thanks in advance,
Dept. og Genetics
Univ. of Copenhagen
At 08.35 04-02-1999 -0800, John Anderson wrote:
>>I have a simple question regarding the behavior of P-
>>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
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