INVERSE PCR

Burkhard Hassel Burkhard.Hassel at t-online.de
Thu Nov 9 13:19:35 EST 2000


On 9 Nov 2000 06:58:28 -0000, obrien at central.murdoch.edu.au ("Philip
O'Brien") wrote:

>We are trying to use inverse PCR to clone the flanking sequences from an
>internal sequenced region in genomic DNA of a fungus.   We have ligated the
>DNA digest into circles  and restricted these to linearize them, but have
>not had any success with getting a discreet product.  Are there any tricks
>to this technique.  What is the best method to rty.
>
>Thanks
>
>phil

Hello!

If you do not know how big the flanking regions are, and if you do not
know the flanking sequence, then you should prepare several digests
with restriction enzymes and circularize them afterwards. The enzymes
shouldn't cut too frequently, otherwise the probability is too high
that they will cut within the target. But they also should not cut to
rarely, because then you would need to amplify very large fragments.
Enzymes like Bgl2 or Hind3 may do.
I don't think linearization (hopefully in between the outward
primers...) have a positive effect.

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Bu.






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