i've done PCR based detection of P elements insertion. My first attempt was
unsuccessful, but I was able to draw some conclusions you might be interested
in. I'd love to hear what other have concluded:
-I was able to detect 1 positive fly in a pool of ~500 (so i played it safe and
pooled up to a 100 flies/tube to grind).
-Too much DNA/RNA/primers will inhibit your PCR reaction (possibly by quenching
*You can pool primers, but i would have more than 4/tube.
*You need RNase in your grinding buffer.
*You should have a Phenol/Chloroform step in your DNA prep (i have a
nice/fast DNA miniprep protocol: 18 samples are in EtOH to ppt in 90
includes RNase and P/C).
-It's true you could get a 7 kb band out of some PCR kits, but I wouldn't count
on it. I play it safe by spreading my primers every 3 kb. Most important is
choosing where on the sequence. Most Ps go into the 5' end of genes, almost
never in coding region. Some genes are cold to Ps (i heard ~50%). Since Ps
tend to hop locally, so if you have a P in your area, you might want to
choose that one...
data compiled from Tower & al '93 (Genetics 133:347), where they used a mini
F2 progeny: 28 800
F2 w/ right genotype: 3600 (male, not Sb delta, and carries a rosy P)
interchromosomal hops: 120
intrachromosomal hops: 36 --->36/3600= 1%
Of those 36 hops:
17 were in a 4.9 kb hotspot (47%)
6 the original P
8 were 0-2 kb away (22%)
3 were 2-3 (8%)
2 were 3-128 (5.6%)
They used PZ (rosy), but you'd be better off with a P marked with white since
you can exclude the Ps that have the same eye color as the parental line
(majority of P stay put).
In fact, if you have a P close to your gene, you might want
to look into male recombination (as Greg so wonderfully described). I tried it
at small scale, and as long as your markers are easy to identify, it's fairly
easy to collect tons of male recombinants. The only problem is to sort out
which ones you want...
About that non-specific primer of yours, just stop using it. Design another one
It'll cost you only ~30$ to get it, you'll save time.
In article <18.104.22.168.19990205091325.0099b800 at biobase.dk>,
lsunicph at BIOBASE.DK (Leif =?iso-8859-1?Q?S=F8ndergaard?=) wrote:
> 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,
> Leif Sondergaard
> 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
> >John Anderson
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