Cloning transgene inertion site

David Micklem dmicklem at cmgm.nospam.invalid
Wed Feb 9 22:45:32 EST 2000


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In article <000d01bf72fb$60598320$2c08d781 at bms.ed.ac.uk>, T. Ian
Simpson \(Holyrood\) <ian.simpson at ed.ac.uk> wrote:

>Hi,
>
>We are tyring to clone the insertion site (5' or 3') of a transgene that we
>have generated and were wondering whether anybody has done this by a method
>that does not involve making a genomic library. We are currently using
>primer extension based approaches, but would greatly appreciate any advice
>from anyone who has succesfully cloned an insertion site or who could point
>us in the direction of a good reference or two,
>
>Many thanks in advance for your expert help !
>
>Best wishes,
>
>Ian.


Inverse PCR works really well at least for Drosophila (or is that what
you meant by 'primer extension based approaches'?).

You can find a detailed protocol here:

http://www.fruitfly.org/methods/

Essentially what you do is design 2 primers for each end of your
transgene, with the primers facing OUTWARD. Cut genomic DNA with a
restriction enzyme that does NOT cut between the two primers. Dilute
the digest (to discourage intermolecular ligation), and circularise by
adding ligase. PCR 'around the circle' with the two primers. Sequence
any bands you get (I've had success sequencing the band directly using
either the same primers or if you have them, nested ones).

At least with flies I've found its worth trying several different
restriction enzymes in parallel. Often one will work well and others
less well, presumably because there is no site near enough to give a
PCR-able sized circle. The 'best' enzyme to use may be one that is
known to cut within the transgene, near the more internal of the two
primers (so that you minimise the amount of transgene that will need to
be PCR'd).

HTH, and feel free to ask if the above makes no sense...

David

-- 
D.R. Micklem,          Time flies like an arrow... Fruit flies like a banana. 
Beckman Center,        Email:dmicklem at cmgm.stanford.edu
Stanford University    Phone: +1 (650) 723-6650
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