Cod UNG

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 electrophoresis of the PCR products revealed UNG reactivation, and thus severe degradation of PCR products of all UNGs tested, except for Cod UNG.

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.

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.

A serial dilution of MS2 viral RNA in human serum was prepared. The mixture was spiked with uracil containing amplicons to mimic carry-over DNA. RNA was quantified using a standard one-step RT-qPCR. The presence of carry-over DNA severely reduced the sensitivity of the assay (red). Inclusion of Cod UNG restored assay sensitivity (green), control sample (grey).

RT-qPCR on human total RNA in presence of Cod UNG, UNG from E. coli, and two competitor cold adapted UNG’s was performed according to protocol. The failure of competitors A and B (cold-adapted UNG’s from marine microorganisms) to fully inactivate at 50-60°C leads to loss of cDNA and the integrity of the PCR reaction.

Cod UNG has a high tolerance for blood components such as serum and anti-coagulants, including EDTA and Na-Heparin.

Cod UNG was tested side-by-side with two commercially available, heat-labile UNG enzymes from cold-adapted marine microorganisms in a typical RT buffer. The enzymes were heat-inactivated at the indicated time and temperatures. Following inactivation, PCR product was added to the samples and incubated for three hours at 37°C. Despite inactivation, upon incubation at 37°C, competitor enzymes degraded DNA (faint bands), while Cod UNG remained inactive in all buffer conditions.