dTTP -> dUTP problems in PCR
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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