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PCR primer design criteria

Andreas Becker becker at ps1515.chemie.uni-marburg.de
Thu Dec 7 13:49:10 EST 1995

Sarven Sabunciyan <sarven at org.ecc.ubc.ca> wrote:


>I have a question about the criteria used to design PCR primers.  I know
>you should avoid hairpin loops, primer dimerization and avoid primers
>which will anneal to other transcripts.  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.



We think that is crucial to avoid A or T at the 3' end. 
Unfortunatly you work with a Mac. For a PC we developed a program that
only creates primer with G or C at the 3' end. If you want even
several C / G's.
It check the whole sequence for repeats in order to avoid this regions
for primer design. Primer ending with the 3' end in these regions make
all lot of misannealing. Our program do avoid this strictly. It is
called PrimerDesign 1.10 

Some general advice for primer designing can be found on our
mitochondria pages. Take a look at my home site.
Andreas Becker

WWW  : http://www.chemie.uni-marburg.de/~becker
eMail: BECKER at ps1515.Chemie.Uni-Marburg.De
Arbeitskreis Prof. Kadenbach, FB Chemie/Biochemie, Hans-
Meerwein-Strasse, Philipps-Universitaet, 35043 Marburg, Germany
Phone: privat +49 6421 47304  Labor +49 6421 28 -5721 Fax -2191

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