Dumb question (?) re cycle sequencing of PCR products

Klaus Salger salger at wap18.zi.biologie.uni-muenchen.de
Wed Nov 9 18:31:18 EST 1994


Guy Hoelzer (hoelzer at unr.edu) wrote:
: 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

: 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.

I don't have any experience in sequencing PCR products but I don't understand
this explanation. As far as I know (which may be not too much)
1. Taq has no 3'-5' exonuclease activity (it produces 3'-overhangs)
   so there should be no degradation at the 3'-end
2. Taq has a 5'-3' exonuclease activity but it is polymerization dependent
3. and this is my most important point - if the primers work in the PCR
   despite the 5'-overhangs, it should also work in the cycle sequencing
   reaction
So, if internal primers give better sequences there has to be another
reason, or am I missing something?
Cheers
  Klaus

--
Klaus Salger                phone : ++49 (0)89 5902 -502
Zoologisches Institut       FAX   :                 -450
AG MacWilliams              e-mail: salger at zi.biologie.uni-muenchen.de
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