The polymerase chain reaction (PCR) can amplify a single molecule over a billionfold. Thus, even minuscule amounts of a contaminant can be amplified and lead to a false positive result. Such contaminants are often products from previous PCR amplifications (carry-over contamination). Therefore, researchers have developed methods to avoid such contamination.
One common strategy is substituting dUTP for dTTP during PCR amplification, to produce uracil-containing DNA (U-DNA). Treating subsequent PCR reaction mixtures with Uracil-DNA Glycosylase (UNG) prior to PCR amplification and subsequent cleavage of apyriminic polynucleotides at elevated temperature (95C) under alkaline conditions (during the initial denaturation step) will remove contaminating U-DNA from the sample (see figure below). This method, of course, requires that all PCR-reactions in the lab have to be carried out with dUTP instead of dTTP.Note the following when using dU-containing PCR products in downstream applications:
The following Roche Applied Science products are suited for preventing carry-over contamination in PCR:
Glycosylase (Cat. No. 1444646)
Uracil-DNA Glycosylase, heat-labile (Cat. No. 1775367)
PCR Core KitPLUS (Cat. No. 1585541)