Dumb question (?) re cycle sequencing of PCR products

Shahram Mori smori at nmsu.edu
Thu Nov 10 00:43:13 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
: > 
: > 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. 
: Guy Hoelzer                                                  

Also, using nested primers allows you to lower the length that you are
PCRing which in turn increases the processitivity of your enzyme. i.e get
full length DNA.
Cheers,

 --
Shahram Mori					   _/\_
Program in Molecular Biology			  _\  /_
Dept. of chemistry and Biochemistry Box 3C	  \_  _/
NMSU  Las Cruces NM				    ||
88003





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