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PCR primer design criteria

Robert Horton horton at biosci.cbs.umn.edu
Fri Dec 8 10:29:00 EST 1995

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	

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