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.

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Heat-labile

Completely and irreversibly inactivated at 55°C
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Contamination control

Ideal for contamination control in RT-PCR, RT-qPCR, qPCR, kPCR and RT-LAMP
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Post-PCR Analysis

Enables post-PCR analysis

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 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.

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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 4ËšC 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.

Chart by Visualizer
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.
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Recommended Protocols

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.

Properties

Specifications

  • 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. 

Stability

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.

Publications

Cod UNG Applications

  1. Presence of bacterial DNA in thrombotic material of patients with myocardial infarction.
    Piñon-Esteban P, Núñez L, Moure R, Marrón-Liñares GM, Flores-Rios X, Aldama-Lopez G, Salgado-Fernandez J, Calviño-Santos R, Rebollal-Leal F, Pan-Lizcano R, Vazquez-Gonzalez N, Bou G, Tomás M, Hermida-Prieto M, Vazquez-Rodriguez JM.
    Sci Rep. 2020; 10, 16299.

  2. LAMP-based ratiometric electrochemical sensing for ultrasensitive detection of Group B Streptococci with improved stability and accuracy.
    Fu Y, Zhou X, Duan X, Liu C, Huang J, Zhang T, Ding S, Min X, A.
    Sensors and Actuators: B. Chemical. 2020; 321:128502

  3. Multiplex Real-Time PCR-shortTUB Assay for Detection of the Mycobacterium tuberculosis Complex in Smear-Negative Clinical Samples with Low Mycobacterial Loads.
    Alcaide F, Trastoy R, Moure R, González-Bardanca M, Ambroa A, López M, Bleriot I, Blasco, L, Fernandez-García L, Tato M, Bou G, Tomás M.
    J Clin Microbiol 2019; 57:e00733-19.

  4. Paper-based Molecular Diagnostics for the Amplification and Detection of Pathogenic Bacteria from Human Whole Blood and Milk Without a Sample Preparation Step.
    Lee JW, Nguyen VD, Seo TS.
    BioChip J. 2019; 13:243-250.

  5. Preamplification with dUTP and Cod UNG Enables Elimination of Contaminating Amplicons.
    Andersson D, Svec D, Pedersen C, Henriksen JR, Ståhlberg A.
    Int. J. Mol. Sci. 2018; 19(10): 3185.

  6. The prognostic efficacy of cell-free DNA hypermethylation in colorectal cancer.
    Ladefoged Rasmussen S; Bygum Krarup H, Gotschalck Sunesen K, Berg Johansen M, Tornby Stende M, Søkilde Pedersen I, Henning Madsen P, Thorlacius-Ussing O.
    Oncotarget. 2018; 9: 7010-7022.

  7. Hypermethylated DNA, a circulating biomarker for colorectal cancer detection.
    Rasmussen SL, Krarup HB, Sunesen KG, Johansen MB, Stender MT, Pedersen IS, Madsen PO, Thorlacius-Ussing O
    PLOS ONE. 2017; 12(7).

  8. Biofilm formation and antibiotic resistance in Klebsiella pneumoniae urinary strains.
    Vuotto C, Longo F, Pascolini C, Donelli G, Balice MP, Libori MF, Tiracchia V, Salvia A and Varaldo PE.
    J Appl Microbiol. 2017; 123: 1003–1018.

  9. Uracil-DNA glycosylase-treated reverse transcription loop-mediated isothermal amplification for rapid detection of avian influenza virus preventing carry-over contamination.
    Kim EM, Jeon HS, Kim JJ, Shin YK, Lee YJ, Yeo SG, Park, CK.
    J Vet Sci 2016; 17 (3), 421-5.

  10. Cell-free DNA promoter hypermethylation in plasma as a diagnostic marker for pancreatic adenocarcinoma.
    Henriksen SD, Madsen PH, Larsen AC, Johansen MB, Drewes AM, Pedersen IS, Krarup H, Thorlacius-Ussing O.
    Clinical Epigenetics. 2016; 8, 117.

  11. Inter- and intraspecific variation in the surface pattern of the dermal bones of two sturgeon species.
    Thieren E, Ottoni C, Popović D, Van Neer W.
    Journal of Applied Ichthyology. 2016; 32(4) 609-628.

  12. A New High-Throughput Approach to Genotype Ancient Human Gastrointestinal Parasites.
    Côté NM, Daligault J, Pruvost M, Bennett EA, Gorgé O, Guimaraes S, Capelli N, Le Bailly M, Geigl EM, Grange T.
    PLoS One. 2016; 11(1): e0146230.

  13. A Concentrated Hydrochloric Acid-based Method for Complete Recovery of DNA from Bone.
    Andersson D, Akrap N, Svec D, Godfrey TE, Kubista M, Landberg G, Ståhlberg A.
    Expert Rev Mol Diagn. 2015; 15(8) 1085-1100.

  14. Properties of targeted preamplification in DNA and cDNA quantification.
    Andersson D, Svec D, Pedersen C, Henriksen JR, Ståhlberg A.
    Expert Rev Mol Diagn. 2015; 15(8) 1085-1100.

  15. Simultaneous elimination of carryover contamination and detection of DNA with uracil-DNA-glycosylase-supplemented loop-mediated isothermal amplification (UDG-LAMP).
    Hsieh K, Mage PL, Csordas AT, Eisentstein M, Soh HT.
    Chem Commu. 2014: 50, 3747.

  16. Complex Species Status for Extinct Moa (Aves: Dinornithiformes) from the Genus Euryapteryx.
    Huynen L and Lambert DM
    PLoS One. 2014; 9(3): e90212.

  17. Highly Informative Ancient DNA ‘Snippets’ for New Zealand Moa.
    McCallum J, Hall S, Lissone I, Anderson J, Huynen L, Lambert DM.
    PLoS One. 2013; 8(1): e50732.

  18. An Efficient Multistrategy DNA Decontamination Procedure of PCR Reagents for Hypersensitive PCR Applications.
    Champlot S, Berthelot C, Pruvost M, Bennett EA, Grange T, Geigl EM.
    PLoS ONE 2010; 5(9): e13042.

  19. Removal of deaminated cytosines and detection of in vivo methylation in ancient DNA.
    Briggs AW, Stenzel U, Meyer M, Krause J, Kircher M, Pääbo S.
    Nucleic Acids Research. 2009; 38(6)e87.

  20. Uracil-DNA glycosylase (UNG) influences the melting temperature (T(m)) of herpes simplex virus (HSV) hybridization probes.
    Leblanc JJ, Pettipas J, Campbell SJ, Davidson RJ, Hatchette TF.
    J Virol Methods. 2008; 151(1): 158-60.

  21. Quantitative assessment of the effect of uracil-DNA glycosylase on amplicon DNA degradation and RNA amplification in reverse transcription-PCR.
    Kleiboeker SB.
    Virology Journal. 2005; 2: 29.

  22. Minimizing DNA contamination by using UNG-coupled quantitative real-time PCR on degraded DNA samples: application to ancient DNA studies.
    Pruvost M, Grange T, Geigl EM.
    BioTechniques. 2005; 38: 569-575.

  23. The structure of uracil-DNA glycosylase from Atlantic cod (Gadus morhua) reveals cold-adaptation features.
    Leiros I, Moe E, Lanes O, Smalas AO, Willassen NP.
    Acta Crystallographica, Section D: Structural Biology. 2004; 59:1357.

  24. Direct isolation of poly(A)+ RNA from 4 M guanidine thiocyanate-lysed cell extracts using locked nucleic acid-oligo(T) capture.
    Jacobsen N, Nielsen PS, Jeffares DC, Eriksen J, Ohlsson H, Arctander P, Kauppinen S.
    Nucleic Acids Research. 2004; 32(7): e64.

  25. Determination of detection and quantification limits for SNP allele frequency estimation in DNA pools using real time PCR.
    Schwarz G, Bäumler S, Block A, Felsenstein FG, Wenzel G.
    Nucleic Acids Research. 2004; 32(3): e24.

  26. A novel real-time PCR assay for quantitative analysis of methylated alleles (QAMA): analysis of the retinoblastoma locus.
    Zeschnigk M, Böhringer S, Price EA, Onadim Z, Maßhöfer L, Lohmann DR.
    Nucleic Acids Research. 2004; 32(16): e125.

  27. Transcripts of developmentally regulated Plasmodium falciparum genes quantified by real-time RT-PCR.
    Blair PL, Witney A, Haynes JD, Moch JK, Carucci DJ, Adams JH.
    Nucleic Acids Research. 2002; 30(10): 2224-2231.

  28. Development of a novel rapid assay to assess the fidelity of DNA double-strand-break repair in human tumour cells.
    Collis SJ, Sangar VK, Tighe A, Roberts SA, Clarke NW, Hendry JH, Margison GP.
    Nucleic Acids Research. 2002; 30(2): E1.

  29. Mutational analysis of the engrailed homeodomain recognition helix by phage display.
    Connolly J, Francklyn C, Augustine JG
    Nucleic Acids Research. 1999; 27(4): 1182-1189.

  30. Relationships between yeast Rad27 and Apn1 in response to apurinic/apyrimidinic (AP) sites in DNA.
    Wu X, Wang Z.
    Nucleic Acids Research. 1999; 27(4): 956-962.

  31. A novel method employing UNG to avoid carry-over contamination in RNA- PCR.
    Udaykumar, Epstein JS, Hewlett IK.
    Nucleic Acids Research. 1993; 21(16): 3917-3918.

  32. Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions.
    Longo M.C., Berninger MS, Hartley JL.
    Gene.1990; 93: 125-128.

  33. Avoiding false positives with PCR.
    Kwok S, Higuchi R.
    Nature. 1989; 339: 237 – 238.

Descriptive Papers

  1. Reduced Hydrophobicity of the Minor Groove Intercalation Loop is Critical for Efficient Catalysis by Cold Adapted Uracil-DNA N-Glycosylase from Atlantic Cod.
    Moe E, Assefa N, Leiros I, Torseth K, Smalås AO and Willassen NP .
    Journal of Thermodynamics & Catalysis. 2015.

  2. Comparative unfolding studies of psychrophilic and mesophilic uracil DNA glycosylase: MD simulations show reduced thermal stability of the cold-adapted enzyme.
    Olufsen M., Brandsdal B.O., Smalås AO.
    Journal of Molecular Graphics and Modelling. 2007: 26(1): 124-134.

  3. Increased flexibility as a strategy for cold adaptation.
    Olufsen M., Smalås AO, Moe E, Brandsdal BO.
    The Journal of Biological Chemistry. 2005; 280: 18042-18048.

  4. Optimisation of the surface electrostatics as a strategy for cold adaptation of uracil-DNAN-glycosylase (UNG) from Atlantic cod.
    Moe E, Leiros I, Riise EK, Olufsen M, Lanes O, Smalås AO, Willassen NP.
    Journal of Molecular Biology. 2004; 343(5): 1221-1230.

  5. 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.

  6. Identification, cloning, and expression of uracil-DNA glycosylase from Atlantic cod (Gadus morhua): characterization and homology modeling of the cold-active catalytic domain.
    Lanes O, Leiros I, Smalås AO, Willassen NP
    Extremophiles. 2002; 6(1):73-86.

  7. Crystallization and preliminary X-ray diffraction analysis of a cold-adapted uracil-DNA glycosylase from Atlantic cod.
    Leiros I, Lanes O, Sundheim O, Helland R, Smalås AO, Willassen NP.
    Acta Crystallographica. 2001; D57: 1706-1708.

  8. Purification and characterization of a cold-adapted uracil-DNA glycosylase from Atlantic cod (Gadus morhua).
    Lanes O, Guddal PH, Gjellesvik DG, Willassen NP.
    Comparative Biochemistry and Physiology – Part B: Biochemistry & Molecular Biology. 2000; 127(3): 399-410.