Likewise, Uracil-N-glycosylase cannot be used in One-Step RT-PCR. But it can be used in Two-Step RT-PCR if dUTP is not used in the RT step. Do some RT mixes use dUTP for reverse transcribing RNA? Or is UNG used to degrade any RNA that failed to get reverse transcribed in RT rxn from being carried over into the qPCR mix?
Some enzymes are inhibited by dU and some are not. Taq pol is not inhibited and hence it can be used in conjunction with dU in the mix. The uridilated PCR product can be degraded before subsequent experiments to prevent carry over. my2c
ABI master mixes contain dUTP and UNG enzyme. The purpose is to degrade any PCR product somehow carried over from reactions. The enzyme works at 50 degrees and then is inactivated by the first 95 degree step. Subsequently, your PCR product will have dU in it. If this contaminates your next PCR run, it will get degraded by UNG and so on. I don't know if this is really a concern if your plates are sealed. You are correct that UNG would kill your cDNA if your RT mix used dUTP. I always use a mix with dTTP, but it's good to check the details.
Uracil-DNA glycosylase can be used to cleave DNA at any site where a deoxyuridylate residue has been incorporated. Because of the heat-lability of Uracil-DNA Glycosylase, heat labile the main application of this enzyme is the prevention of carryover contamination in PCR. In contrast to the enzyme from E.coli; using this preparation of UNG it is not necessary to freeze the PCR product immediately after amplification or to hold the reaction mixture at +70 °C.
For completeness, let me add that RNA serves as a template for the RT enzyme to copy as DNA, but it isn't degraded in the process. RNA may remain in a duplex with the newly made DNA. There is some evidence that this can interfere with subsequent PCR, which is why people may treat with RNAseH after the RT step. Whether this is important seems to depend on the particular transcript to be amplified. Free RNA can't serve as template for Taq, so its not as much of a concern.
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