AmpErase™ Uracil N-Glycosylase (UNG) - FAQs

AmpErase UNG (Uracil N-glycosylase) is an enzyme utilized in a powerful method for elimination of carryover PCR products in Real-Time PCR. This method modifies PCR products such that in a new reaction, any residual products from previous PCR amplifications will be digested and prevented from amplifying, but the true DNA templates will be unaffected.

Here is how it works: During amplification, dUTP is substituted for dTTP, resulting in dUTP-containing amplicons. In subsequent reactions, a short pre-PCR incubation step will allow the AmpErase UNG to digest any dUTP containing DNA. Since AmpErase UNG is active on both single- and double-stranded dUTP-containing DNA, the procedure should work on dU-containing PCR products from standard or asymmetric PCR amplifications. However, uracil ribonucleotide residues in RNA, novel DNA containing dTTP, or cDNA containing dTTP will not be suitable substrates for UNG, so your templates will be unaffected.

Note that this is a proactive method to prevent contamination from future reactions, but will not help with a pre-existing contamination problem of standard dTTP-containing PCR products. That can only be remedied with thorough cleaning of lab surfaces, equipment and air filters.

PCR products containing dUTP residues are generally equivalent to dT-containing PCR products as hybridization targets and as templates for dideoxy-terminated sequencing reactions. They can also work equivalently as targets for direct cloning as long as they are transferred into UNG-minus bacterial hosts.

The recognition of dU-containing DNA by restriction endonucleases has been studied, and depending on the specific endonuclease there may be no effect on enzymatic activity on the substitution of dU for dT (e.g., EcoR1 and BamH1), or the dU-containing DNA is cleaved more slowly than dT-containing DNA (e.g., Hpa1, HindII, and HindIII). The most accurate answer on the effects of substituting dU for dT are best determined using your own empirical studies.