Increase PCR specificity W/ DMSO?

PC386 at TAUNOS.BITNET PC386 at TAUNOS.BITNET
Thu Jan 30 22:29:15 EST 1992


Dear NetMates.

Sandra Pena de Ortiz  from the University of Cincinnati asks :

>Could anyone comment about how to increase primer specificities in PCR.
> I have heard or read a litte bit about formamide and DMSO.
> Thanks for any information.
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Since I belive it may benefit more than one ...     - here it comes !

The basic idea of bringing PCR to be highly specific is the concept of
being minimalists.  i.e. - Run it with the highest stringency you can.
* Some of the primary demamnds - Take extra caution when you plan your
primers. One can align the template sequence on the computer with databanks
sequences. Following that you can choose areas of minimal homology.
 Choose a primer couple with close enough Tm's . Mg++ concentrations must
be titrated between 1mM - 10mM to obtain best results. (This is for every
new primer).

* You may try the "Hot Start" procedure. i.e.- Prepare a separate mix of
Taq Polymerase with 1X buffer & BSA, in a volume of ~10 microlit./RXN.
Start the cycles without Taq. Primary denaturation, anneal and let it go to
extension (72 C) , Then press the "Hold" at 72 C , and add Taq (Through the
oil...) .  It worked superb in my hands.

* Reduce dNTP's concentration to ~ 40uMole/RXN , this helps a lot to reduce
mispriming (dNTP's also bind some of the Mg++).

* Reduce primer concentration. (even down to 10 pMole... but normally to ~
50 - 100).   -- VERY important when you face problems.

* You can use 5% - 10% of Formamide or better - DMSO. This opens up
secondary structures and (NOTE !!!) elevates the Tm of the primers and the
product.  In my hands, Formamide didn't do enything, while DMSO actually
saved my life ....  ;  Note-  DMSO reduces Taq activity by 50%, but when it
helps - it worths it...

* If you have problems of reach GC sequences, you may try to use a Cytidine
analog instead of dCTP . In a neighboring Lab it worked fantastic.


I have tryed all of the above (except the last), and warmly reccomend...

Hope it helps,   and will be happy to know if it did.

Yours,

*****************************************************************
* Benny Shomer                                                  *
* Tel-Aviv University                                           *
* Sackler School of Medicine, Dept. of Embryology and Teratology*
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* Snail  :  Sheba Medical Center, Tel-Hashomer, 52625,   Israel.*
* E-mail :    PC386 at VE.TAU.AC.IL                                *
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*                                     (c/o Dr. R. Ehrlich)      *
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