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

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 LJNGs 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 RNAin human serum was prepared. The mixture was spiked with uracilcontaining amplicons to mimic carry-over DNA. RNA was quantifiedusing a standard one-step RT-qPCR. The presence of carry-overDNA severely reduced the sensitivity of the assay (red). Inclusion ofCod 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 coldadapted UNG’s was performed according to protocol. The failureof competitors A and B (cold-adapted UNG’s from marine microorganisms) to fully inactivate at 50-60°C leads to loss of cDNA andthe integrity of the PCR reaction.

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

Cod UNG was testedside-by-side two commercially available, heat-labile UNG enzymes from cold-adapted marine microorganisms in a typical RTbuffer. The enzymes were heat-inactivated at the indicated timeand temperatures. Following inactivation, PCR product was addedto the samples and incubated for three hours at 37°C. Despite inactivation, upon incubation at 37°C, competitor enzymes degradedDNA (faint bands), while Cod UNG remained inactive in all bufferconditions.