Cod Uracil-DNA Glycosylase
Cod Uracil-DNA Glycosylase (Cod UNG) from Atlantic cod 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.
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 completely and irreversibly inactivated by heat thus ensuring that sample integrity is maintained long-term regardless of storage conditions.
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.
1. Contamination control in PCR, qPCR and one-step RT-qPCR
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 0.2 U Cod UNG directly to your 20 µl PCR reaction.
- Pre-incubate for 5 min at room temperature.
- For RT-qPCR, reverse transcribe your RNA at 50-55°C.
- Run your PCR.
Store your PCR product at -20°C or 4°C degrees.
2. Contamination control in RT-LAMP
Cod UNG is ideal for contamination control in RT-LAMP. One unit of Cod UNG per 30 μl reaction is sufficient for removing even high concentrations of carry-over contamination.
- Ensure that you use dNTP mixes containing dUTP in your experiments.
- Check that the RT-LAMP reaction is compatible with dUTP by running side-by-side reactions containing different rations of dUTP to dUTP (100% dUTP, 90% dUTP, 80% dUTP and 0% dUTP).
- Add 1 U Cod UNG directly to your 30 µl RT-LAMP reaction.
- Prepare the reaction mix on ice.
- Analyze your RNA at 65°C, no preincubation is necessary.
Please refer to Protocol.
- Unit definition: One Unit is defined as the amount of enzyme that liberates 1 nmol Uracil per hour from Uracil-labelled DNA at 37°C in 70 mm Tris-HCI pH 8.0 (@25°C), 10 mM NaCI, 1 mM EDTA and 0.1 mg/ml BSA.
- Specific activity: > 500 000 Units/mg.
- Purity: Purified to apparent homogeneity by SDS-PAGE. No nuclease activity is detected.
- Concentration: Minimum 1 000 Units /ml.
Minimum shelf life for standard formulation at -20°C is 3 years. In practice we find that storage at 4°C is possible for at least 6 months. The enzyme activity is also preserved upon multiple freeze-thaw cycles.
Cod UNG Applications
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- Reduced Hydrophobicity of the Minor Groove Intercalation Loop is Critical for Efficient Catalysis by Cold Adapted Uracil-DNA N-Glycosylase from Atlantic Cod.
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- Comparative unfolding studies of psychrophilic and mesophilic uracil DNA glycosylase: MD simulations show reduced thermal stability of the cold-adapted enzyme.
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- Increased flexibility as a strategy for cold adaptation.
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- Optimisation of the surface electrostatics as a strategy for cold adaptation of uracil-DNAN-glycosylase (UNG) from Atlantic cod.
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Journal of Molecular Biology. 2004; 343(5): 1221-1230.
- The crystal structure of Uracil-DNA N-glycosylase from Atlantic cod (Gadus morhua) reveals cold-adapted features.
Leiros I, Moe E, Lanes O, Smalås AO, Willassen NP.
Acta Crystallographica. 2003; D59: 1357-1365.
- Identification, cloning, and expression of uracil-DNA glycosylase from Atlantic cod (Gadus morhua): characterization and homology modeling of the cold-active catalytic domain.
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- Crystallization and preliminary X-ray diffraction analysis of a cold-adapted uracil-DNA glycosylase from Atlantic cod.
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- Purification and characterization of a cold-adapted uracil-DNA glycosylase from Atlantic cod (Gadus morhua).
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