Sarven Sabunciyan (sarven at org.ecc.ubc.ca) wrote:
: ...I've also read (and been told)
: that a primer should not have a T at the 3 prime end since this will
: result in non-specific annealing. I've been asking for demo versions of
: commercially available primer design packages as well as using shareware
: programs (by the way, I am working on a Mac). Several of these programs
: have given me primers with T's at the 3 prime end. In fact when I asked
: a friend who uses Oligo to come up with primers, Oligo gave a primer with
: a T at the end (granted it was an older version of the program). Is a T
: at the 3 prime end of a primer not all that crutial for specific PCR or
: are these programs not all that reliable? Can somebody give me
: references about designing primers? Any help is much appreciated.
A "T" at the end may not work as well if you are trying to use a
single base mismatch to distinguish alleles. I don't think it is
a big problem otherwise.
Incidentally, the primer:template mismatches A:A, A:G, C:C, or
G:A are reported to be particularly effective at inhibiting
priming on the mismatched template. See:
Kwok, S. Kellogg, D.E., McKinney, N., Spasic, D.,
Goda, L., Levenson, C., and Sninsky, J.J. Effects
of primer-template mismatches on the polymerase
chain reaction: human immunodeficiency virus type 1
model studies. Nucleic Acids Research. 18:999-1005,
Huang, M.M., Arnheim, N., and Goodman, M.F.
Extension of base mispairs by Taq DNA polymerase:
implications for single nucleotide discrimination
in PCR. Nucleic Acids Research. 20:4567-73, 1992.
Bob Horton horton at biosci.cbs.umn.edu
Department of Dermatology (!?!)
University of Minnesota Medical School *CAUTION*
Minneapolis, MN 55455 USA .sig under construction