Function of dUTP in real-time PCR?

Duncan Clark blackhole at abuse.plus.com
Wed Oct 27 10:44:01 EST 2004


Historians believe that in newspost 
<vuymzy8fakz.fsf at soggy-fibers.csail.mit.edu> on Wed, 27 Oct 2004, Tom 
Knight <tk at csail.mit.edu> penned the following literary masterpiece:
>The final
>amplification product will contain dUTP, and will be a poor template
>for the next PCR reaction.

Providing Uracil-N-glysolyase is present in that next PCR reaction.

A dUTP containing substrate template will normally PCR fine. It's the 
IUNG that makes the difference.

UNG is used for an initial 2mins at 50C then PCR as normal.

Beware though that although it is normally E.coli UNG that is used, some 
does survive PCR. That small amount is sufficient to degrade the 
amplified PCR product even at 25C. Not a problem if it is a real-time 
PCR product (providing you have run any SYBR Green I melt curve 
immediately following the amplification) but don't do it if you want the 
PCR product afterwards.

Duncan
-- 
The problem with the gene pool is that there is no lifeguard.

Duncan Clark
GeneSys Ltd.



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