Primer extension using Sequencers

Patrick Lynch
Fri Apr 27 04:46:26 EST 2001


Hi David,

I have carried out a specific type of primer extension called GMPD, which is
used to detect mutations, on an old ABI 373 using end labelled primers.  The
normal and mutant alleles caused the primer to be extended by 3 and 7 bases
respectively - therefore, I had to resolve a 28 base DNA molecule from a 24
base extensions as well as free 21 base primer.  This worked well on a 8%
polyacrlyamide gel (7M urea 1X TBE).  The only problem was interference from
unextended (free) primer - it's very hard to get a homogeneous primer band -
there's always lots of extra bands (from the synthesis of the primer) that,
if bad enough, can obscure the primer extensions.  Therefore, only use
fluorescent primers that are cleaned up after synthesis (even these can be
dirty) but the best (and cheapest way!) is purify the oligo yourself by
running a large portion of your oligo out on a preparative denaturing gel
and cut out and purify the oligo (it's in Maniatis).  One other trick to
remove free primer after extension is to use ExoI (as long as your extension
products are not affected).

Hope this helps.

Patrick


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