dTTP -> dUTP problems in PCR

John Ladasky jladasky at pmgm.Stanford.EDU
Wed Mar 4 12:23:24 EST 1998


In article <01bd4778$be177d60$6096f686 at ifremer.ifremer.fr>,
Richard THIERY <rthiery at ifremer.fr> wrote:
>
>
>Nico van Belzen <belzen at hema.fgg.eur.nl> a écrit dans l'article
><6dja4b$r8$4 at popeye.eur.nl>...
>> Hi all, 
>> 
>> (posting this for a colleague) 
>> In order to prevent contamination, I want to use dUTP/UDG (uracyl 
>> glycosylase) in all diagnostic PCR systems. However, sometimes the 
>> reaction is not working when I change from dTTP to dUTP. I tried to 
>> increase the dUTP concentration, this did not help. Trying other 
>> brands of Taq polymerase does not work. Does somebody know a solution 
>> for this problem? 
>> 
>> Best regards, 
>> 
>If you are using UDG before doing the PCR reaction make sure that you
>inactivate properly this enzyme (10 min @ 95°C), otherwise any residual
>activity will destroy your PCR products. Also it is better if the annealing
>temperature of your primer pair is above 60°C. Try also adding an extra 5
>cycles to your PCR because you may have a decrease in PCR efficiency any
>way (eg 40 cycles instead of 35 cycles).
>Hope this helps, good luck.

	What exactly is being amplified?  dUTP is known to decrease yields,
or even kill reactions, when one is amplifying longer products (say >1kB).
dUTP was (is?) a common contaminant/side product in deoxynucleotide tri-
phosphate preparations.  We had some reactions that would not work, and 
eventually we called the manufacturer of the dNTP's.  They sent us a bullet-
in that described the problem and how they had corrected it in their new
lots of dNTP.  And, by the way, the new nucelotides worked fine.

-- 
Rainforest laid low.
"Wake up and smell the ozone,"
Says man with chainsaw.					- John Ladasky



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