Inverse PCR -- degraded DNA ends?

John Ladasky jladasky at pmgm.Stanford.EDU
Thu Jan 29 19:04:53 EST 1998


Greetings, everyone,

	I have been using inverse PCR to clone sequences from mammalian
genomic DNA.  After much frustration, I finally got it to work.  (Burk-
hard Hassel, if you're reading this, thanks for the protocol!)  However,
I have noticed something unusual.  On two separate occasions, looking at
two different genes and using two different restriction enzymes, I have 
obtained clones which lack the restriction site that should be present
somewhere in the middle of the clone!  In one case, I was able to figure
out where the breakpoint was, to within about 10 bp, because I had a
homolgous sequence against which to align my inverse PCR product.  In the
second case, I was reading a completely novel sequence.  So once I fin-
ished, I tried PCR against genomic DNA to see which fragments would amp-
lify.  This allowed me to locate the missing restriction site to within a
few hundred bases, out of about 2 kB.  But I would like to do better than
that.

	Presumably, the reason that my restriction sites are missing is
that, during the cutting and/or ligation steps, the ragged ends of the DNA
are getting degraded.  How the DNA then manages to ligate, I don't know.
However, I am able to reamplify the same fragments from the ligated DNA
several times, and I get the same sequence.  So whatever degradation is
occurring is not random!  Has anyone had similar difficulties with the ends
of their inverse PCR products?  How would you recommend solving them?

-- 
Rainforest laid low.
"Wake up and smell the ozone,"
Says man with chainsaw.					- John Ladasky



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