PCR/single 3' mismatch

Shahram Mori smori at nmsu.edu
Fri Nov 11 22:55:38 EST 1994


kenneth garson (cancer research) (kgarson at labsun1.med.uottawa.ca) wrote:

: I have a PCR template which is degenerate at at least one position. I
: would like to amplify specifically those templates which contain a G at
: this position. If I design a PCR primer whose 3' base matches a "G" at
: this degenerate site, could I be assured of only amplifying templates
: which contain a "G" at this position, or will annealed primers with a
: single 3' mismatch have a probability of amplifying? Cloning is not really
: a favourable option since the amount of available template is low, so PCR
: is necessary, and the percentage of template harbouring a G at this site
: may be low so screening clones of PCR products using external primers
: would be  a lot more work.  Any ideas? Any references?

: Thank you for any input.


: Ken Garson
: Ottawa Centre for Cancer Research
: University of Ottawa,
: Ottawa, Ontario
: Canada
: K1H 8M5
: kgarson at labsun1.med.uottawa.ca

Dear Ken,
having one mismatch at the 3' end is not that important. Taq, in fact, still
amplifies with sequences with 3' ends rich in G and T mismatches. I am
using this fact for DDRT-PCR. I guess your best bet is to increase the
annealing tempertaure to VERY high in order to discriminate for the single
base. 
I hope this helps
Cheers,

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