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Function of dUTP in real-time PCR?

Function of dUTP in real-time PCR? - Protocols and Methods Forum

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  #1  
Old 10-27-2004, 11:27 AM
Paul Wary
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Default Function of dUTP in real-time PCR?



Hello,

I read in a description of mixes used for real-time PCR that they
contain dUTP "to prevent re-amplification of carryover PCR products".
Can anyone tell me how this works or give me a reference?
Thanks in advance.

Paul
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  #2  
Old 10-27-2004, 03:24 PM
Tom Knight
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Default Function of dUTP in real-time PCR?

[Only registered users see links. ] (Paul Wary) writes:

The mix also contains an enzyme which selectively degrades dUTP
containing DNA. This enzyme is degraded early in the PCR process.
Carryover PCR product will have dUTP and will be degraded. Real
template will have dTTP, and will not be degraded. The final
amplification product will contain dUTP, and will be a poor template
for the next PCR reaction.


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  #3  
Old 10-27-2004, 03:44 PM
Duncan Clark
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Default Function of dUTP in real-time PCR?

Historians believe that in newspost
<[Only registered users see links. ].edu> on Wed, 27 Oct 2004, Tom
Knight <[Only registered users see links. ].edu> penned the following literary masterpiece:

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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  #4  
Old 10-27-2004, 03:54 PM
salanti
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Default Function of dUTP in real-time PCR?


:

Hi.
I have asked before, but did not get some "sure" answers! Is it possible to
TA Topo clone directly from a real time PCR reaction mix containg UTPs (and
sybr green). I have tried a few times but with limited succes (ended up
doing a normal PCR and cloned from that)
Thanks
Salanti


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  #5  
Old 10-27-2004, 04:53 PM
Duncan Clark
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Default Function of dUTP in real-time PCR?

Historians believe that in newspost <%wPfd.2365$[Only registered users see links. ]>
on Wed, 27 Oct 2004, salanti <[Only registered users see links. ]> penned the following
literary masterpiece:

If your E.coli is a standard cloning strain then I would expect the host
UNG to degrade the TA insert. Not necessarily all but enough to reduce
your cloning efficiency. Given that one normally sees say a hundred or
so colonies on a TA plate the host UNG is probably efficient enough to
mean you won't see the clone you are looking for on that plate.

UNG and dUTP is after all the basis for Kunkel mutagenesis.

Duncan
--
I love deadlines. I especially like the whooshing noise they make as
they go flying by.

Duncan Clark
GeneSys Ltd.
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  #6  
Old 10-29-2004, 02:43 PM
Paul Wary
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Default Function of dUTP in real-time PCR?

Tom Knight wrote:

Ummm, where do I actually get any carryover PCR product from? I use
real-time PCR for quantification of gene expression levels, so I
reverse transcribe mRNA to cDNA and use that cDNA for the real-time
PCR. I can't see where I get that carryover PCR product from.

Paul
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  #7  
Old 10-29-2004, 03:10 PM
Tom Knight
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Default Function of dUTP in real-time PCR?

[Only registered users see links. ] (Paul Wary) writes:

Well, that's the point. You're *not* supposed to get carryover. But,
because in principle PCR can amplify a single copy of a gene, it is
difficult keep reagents, benchtops, pipets, and air free of copies of
the gene you are interested in. Yesterday you amplified 10**15 copies
of that gene. Some probably ended up on your bench. Some of those
probably ended up in the tube you are PCRing today. You would like
those not to be templates.


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  #8  
Old 11-01-2004, 08:58 PM
Dag Rune Gjellesvik
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Default Function of dUTP in real-time PCR?


"Paul Wary" <[Only registered users see links. ]> skrev i melding
news:[Only registered users see links. ] m...

Hello,
Well, if you use the same amplification primers, you still will amplify any
PCR products along with your cDNA. And you won't know if your amplicon came
from cDNA or PCR product. Maybe one or two extra template molecules doesn't
matter too much in gene expression studies - it's worse if you want to know
the presence of HIV virions, though.

Dag Rune Gjellesvik
Biotec Pharmacon ASA


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