dUTPs in qRTPCR rxn - (Jan/07/2010 )
I'm currently trying to educate myself in using qRTPCR to check for knockdown efficiency in a cell line. I'm using a one-step sybr green kit, and I noticed that the dNTPs include dUTP in lieu of dTTPs. This protocol does not recommend the use of uracil DNA glycoylase (UDG) to eliminate carryover nucleic acid, so why the dUTPs?
And this brings me to a question about how UDG carryover prevention works, in general. If you substitute dUTPs for dTTPs in your reaction, and then treat with UDG, won't you degrade the DNA product that you want to analyze? What would be a "carryover prevention scenerio"?
the UDG enzyme is active before the heat activation of the polymerase and gets inactivated during this heating phase. the principle is, that if you are incorporating UTPs instead of TTPs you can eliminate contaminations caused by PCR products from previously performed PCR reactions but of course not from sample cross contamination or plasmid.