Dumb question (?) re cycle sequencing of PCR products

Guy Hoelzer hoelzer at unr.edu
Tue Nov 8 15:57:51 EST 1994


In article <39nkfe$8ab at mserv1.dl.ac.uk>, (Dave Johnston)
<daj at mailserver.nhm.ac.uk> wrote:

> Hi,
> When cycle sequencing PCR products, the dogma seems to be that you can't 
> use either of the original PCR primers as a sequencing primer. Why not? 
> They bind and act as primers for second strand synthesis by Taq (or the PCR 
> wouldn't work in the first place), what more do you need? Is it just a 
> scam to get us to buy more oligos or am I missing something?
> 
> Thanks
> DAJ
> 
> David A. Johnston
> Research Fellow,
> Dept of Zoology, The Natural History Museum, Cromwell Road,
> South Kensington, London SW7 5DB. England
> (tel 071 9389297, fax 071 9388754, email daj at nhm.ac.uk)

This is not such a dumb question.  Your logic is correct and you can use
PCR primers to sequence your PCR product.  However, you are better off
using an internal primer, which can overlap substantially one of your
original PCR primers.  I believe the reason for this is that most Taq has
some sort of exonuclease activity that tends to degrade the ends of your
DNA molecules.  Therefore the 5' end of your oligo may be hanging off the
template and the melting temperature of that primer will effectively be
lowered.  Choosing a sequencing primer that starts at least several bases
toward the interior of your PCR fragment will avoid this problem and
produce a stronger sequence signal.
-- 
******************************************************************************
Guy Hoelzer                                                  
hoelzer at unr.edu
Dept. of Biology
University of Nevada Reno
Reno, NV  89557
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