Cod Uracil-DNA Glycosylase (Cod UNG) from Atlantic cod is the only commercially available UNG enzyme that is completely and irreversibly inactivated by moderate heat treatment. The enzyme is produced in a recombinant E. coli (ung–) strain that contains a modified Cod UNG gene.

There are several commercially available Uracil-DNA glycosylases on the market today. Most of them are of bacterial origin and work well if you have no intention to further analyze the PCR products post-PCR. However, if you want to store your PCR products for downstream analysis such as cloning and sequencing, the reactivation of UNG and subsequent degradation of your PCR products are a problem with most of the commercially available UNGs. Cod UNG from ArcticZymes is the only commercially available UNG today which is completely and irreversibly inactivated by heat.

Figure 1. The only UNG that become completely and irreversibly heat-inactivated is Cod UNG.

This is illustrated in figure 1, where various UNGs were tested for residual activity after heat inactivation. PCR was performed with dUTP and 1 Unit of 5 different commercially available UNGs. Post-PCR, the PCR products were incubated at room temperature for various time intervals, followed by heating and subsequent cooling. Gel electrophoreses of the PCR products revealed UNG reactivation, and thus severe degradation of PCR products of all UNGs tested, except for Cod UNG.

Figure 2. Chromatograms of sequenced PCR products pre-treated with 1 U Cod UNG (A) or 1 U E.coli UNG (B) and incubated at room temperature for 3 hours. Only Cod UNG leaves sequence quality intact.

Post-PCR sequence quality and integrity were further evaluated by sequencing the PCR products. PCR was performed with one of four different commercially available UNGs added to the mastermix. Post-PCR, the PCR products were incubated at room temperature or 4˚C at various time intervals. Samples were subsequently purified and sequenced. Sequence data were thoroughly analyzed with emphasis on reduced sequence quality as a result of UNG reactivation. As illustrated in both figure 2 and figure 3, samples treated with UNG showed severe degradation of PCR products due to UNG reactivation, except for samples treated with Cod UNG.

Figure 3. UNG reactivation resulted in degraded sequences. Sequence data of PCR products pretreated with various UNGs and incubated at room temperature. All samples, except samples incubated with Cod UNG, demonstrated reactivation of UNG and severe degradation of PCR products.