[Q] primer design

Curtis R. Altmann altmanc at rockvax.rockefeller.edu
Wed Apr 1 12:39:28 EST 1998


I would have to say that the second primer is better however neither
primer is very good.  This is due to the presence of the g's.  The G run
will result in the formation of triplex structures which are quite stable
and will interfere with most enzymatic reactions.  If you simply have
selected G's as an example and the sequence is not a homopolymer, then the
first primer is better.  There are significant energy costs to break the
helix and extend a region of non homology.  In the case of the first primer,
your Tm would be the total calculated for the full homology.  For the
second, the Tm would be closer to the Tm for the two regions of homology
with some increase in stability due to the fact that once one of the regions
hybridized, the other would be constrained.  For PCR purposes, the 3' end is
the most 
important region for homology, the 5' does not require any homology

Hope this is of some use,
Curtis R. Altmann, Ph.D.
The Rockefeller University
----------
In article <Pine.SGI.3.96.980401131200.932A-100000 at ccs.sogang.ac.kr>, "Kim,
Cheon" <s197156 at ccs.sogang.ac.kr> wrote:


>
>Hi, netters.
>
>Could you tell me which primer is better?
>
>1.The primer which has a 30 base pair homology to the template 
>  but a 40 mer random base at its terminus. looks like below. 
>
>  5'- a
>        t
>          c 
>            gggggggggggggg-3' : primer
>            cccccccccccccc    : template
>
>
>2. The primer that has two, 15 base pair homology at it's termini
>   but a 40 mer randome base in the middle of it. looks like below.
>
>           atcgatcgact
>  5'-gggggg           gggggg-3' : primer
>     ccccccccccccccccccccccc    : template
>
>
>
>thanx in advance...
>
>
>
>
>===========================================================================
====
> 
>          Judge a man by his questions, rather than his answers.
>
>===========================================================================
====
>
>





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