Cod Uracil-DNA Glycosylase

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.
Image

High robustness

Heat-labile, completely and irreversibly inactivated at 55°C
Image

Contamination control

Ideal for contamination control in RT-PCR, RT-qPCR, qPCR and kPCR
Image

Post-PCR Analysis

Enables post-PCR analysis

The only true heat-labile Uracil-DNA Glycosylase

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 showing the only UNG that become completely and irreversibly heat-inactivated is Cod UNG

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.

Image

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.

Recommended Protocol

PCR

Cod UNG works in all commercially available master mixes. Be sure that you have used dUTP containing dNTP mixes in your previous PCR experiments.

  • Add 0.25 U Cod UNG directly to your 25 µl PCR reaction
  • Pre-incubate for 5 min at room temperature
  • Run your PCR

Store your PCR product at -20°C or 4°C degrees for as long you want, before analysis.

One-step RT-PCR

  • Add 0.2 U Cod UNG directly to your 20 µl RT-PCR reaction
  • Preincubate at room temperature for 5 min
  • Reverse transcribe your RNA at 50- 55°C
  • Run your PCR

Properties

Cod UNG works in all commercially available master mixes. Be sure that you have used dUTP containing dNTP mixes in your previous PCR experiments.

  • Add 0.25 U Cod UNG directly to your 25 µl PCR reaction
  • Pre-incubate for 5 min at room temperature
  • Run your PCR

Store your PCR product at -20°C or 4°C degrees for as long you want, before analysis.