Sequencing PCR product ???

Brain Foley brianf at med.uvm.edu
Mon Nov 8 13:11:38 EST 1993


Ali Karami (ali at biobase.aau.dk) wrote:
:  Dose anybody hase  any  experience Sequencing PCR product directly 
:  Please send me your comments .

:  thank you 

:  ali karami 
: Inst of Medical Microbiology
: Copenhagen University
: ali at biobase.aau.dk

	I have quite a bit of experience, and there is also a LOT of
information that has been posted to this list over the last two years.
In short, one could write a whole book on sequencing PCR products.
I don't have time to do so right now...
	What type of info do you want?  Single-standed PCR product or
double standed?  PCR product from homozygote or herterozygote (i.e. is
the product homogeneous or does it contain different sequences?)?  Do
you want to really sequence the whole PCR product, or just confirm a 
small region of sequence?  DO you want to use end-labelled primers or
incorporate radiolabel during elongation?

	I suggest you look at all the information that has already
been posted to this list (point your gopher at the IUBIO archives)
and also look at a few issues of BioTechniques that deal with this
topic.
	In my experience, directly sequencing a PCR product is a very
powerful and useful technique.  But it is not a quick-and-easy way
to sequence.  You will likely spend a lot of time trouble shooting
before you start to produce readable sequencing gels.  

--
********************************************************************
*  Brian Foley               *     If we knew what we were doing   *
*  Molecular Genetics Dept.  *     it wouldn't be called research  *
*  University of Vermont     *                                     *



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