Preventing Carry-over Contamination With Uracil-DNA Glycosylase

Preventing Carry-over Contamination with Uracil-DNA Glycosylase

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 (95°C) 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:

• PCR products containing dU perform as well as those containing dT when used as hybridization targets or as templates for dideoxy sequencing.

• PCR products containing dU can be cloned directly, if they are transformed into ung–bacterial hosts.

• A dU-containing substrate is readily digested by some common restriction enzymes (e.g. Eco RI and Bam HI), while others show reduced activity (e.g. Hpa I, Hind II, Hind III) on these substrates.

• We do not recommend the use of dU-containing DNA for protein binding or DNA-protein interaction studies.

The following Roche Applied Science products are suited for preventing carry-over contamination in PCR:

Uracil-DNA Glycosylase (Cat. No. 1444646)
Uracil-DNA Glycosylase, heat-labile (Cat. No. 1775367)
PCR Core Kit^PLUS (Cat. No. 1585541)