What's the function of UNG in qPCR? - (Feb/27/2006 )

qPCR kits contain dUTP and UNG to prevent carry over from previous qPCRs. What is the mechanism by which it degrades the contaminating DNA but not the DNA of our interest. I would be very glad if someone explain me the mechanism. Thanx in advance.

-bhaskar_dutta-

HI
Maybe this is what you're searching for ? :
Minimizing DNA contamination by using UNG-coupled quantitative real-time PCR on degraded DNA samples: application to ancient DNA studies

by Mélanie Pruvost, Thierry Grange, and Eva-Maria Geigl

-fred_33-

Look at this site:

http://www.bio.umass.edu/biology/genomics/...0in%20qPCR'

-batsirayi-

QUOTE (batsirayi @ Mar 3 2006, 10:32 AM)

Look at this site:

http://www.bio.umass.edu/biology/genomics/...0in%20qPCR'

This is the info from the link above

UNG stands for Uracil-N-glycosylase. It is an enzyme, which hydrolyses uracil-glycosidic

bonds in DNA containing dUTP, therefore degrading the DNA into small fragments. In this

way contamination from previous qPCR reactions can be avoided.

My question:
Do we include dUTP in our real time mix...I thought it was dTTP anyway I still couldnot get how UNG will not digest new DNA that are synthesized

-BusyBee-

Your PCR cycle should have a 10 min 95°C deactivation of UNG step before the denaturing and annealing/extension cycles.

-soluene-

QUOTE (soluene @ Mar 8 2006, 08:30 PM)

Your PCR cycle should have a 10 min 95°C deactivation of UNG step before the denaturing and annealing/extension cycles.

The problem with most UNG enzymes is that even if they are inactivated, they will reactivate after a while in the fridge. That's why 10 min at 95°C could be usefull.
10 min at 95°C is a hard treatment, which is not always a good idea.

If you use Cod UNG you don't need to worry about deactivation and reactivation. This enzyme has a halflife of 30 sec. at 50°C, while 10 min dectivation at 60°C is suggested, and no reactivation have ever been seen with this enzyme.

-Gerd-

ok picture this:

you have a DNA template.
You do a PCR with it, with normal dNTPs and also some dUTPs.
You then do whatever you want with the amplicon, which has dUTPs in it.

Now lets say some of those amplicons are floating around and can contaminate your next sample.
So then you add some UNG to your PCR and you change the thermal cycling conditions...
- you activate your UNG: UNG will now go and degrade any DNA with dUTP in it. right now the only DNA with dUTP in it is your contaminate: your actual amplicon of interest has yet to be amplified.
- you inactivate your UNG - no more degradation of anything.
- then you do normal thermal cycling, incorporating dUTP into your present amplicon of interest.

then next time you do something else, the UNG will degrade the last PCR product but not the one you are interested in because it hasn't been made yet.

hope that makes sense

-kcyho-