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.
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.
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.
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.|
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.
- 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
- Unit definition: One Unit will liberate 1 nmol Uracil from Uracil-containing DNA per hour at 37°C.
- 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 at -20°C is 2 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.
- Reduced Hydrophobicity of the Minor Groove Intercalation Loop is Critical for Efficient Catalysis by Cold Adapted Uracil-DNA N-Glycosylase from Atlantic Cod
Elin Moe, Netsanet Gizaw Assefa, Ingar Leiros, Kathrin Torseth, Arne O Smalås and Nils Peder Willassen
Journal of Thermodynamics & Catalysis
- Purification and characterization of a cold-adapted uracil-DNA glycosylase from Atlantic cod.
Lanes O., et al. (2000)
Comparative Biochemistry and Physiology – Part B: Biochemistry & Molecular Biology. 127: 399-410.
- 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., et al. (2002)
Extremophiles. 6: 73-86.
- Crystallization and preliminary X-ray diffraction analysis of a cold-adapted uracil-DNA glycosylase from Atlantic cod.
Leiros I., et al. (2001)
Acta Crystallographica. D57: 1706-1708.
- The crystal structure of Uracil-DNA N-glycosylase from Atlantic cod (Gadus morhua) reveals cold-adapted features.
Leiros I., et al. (2003)
Acta Crystallographica. D59: 1357-1365.
- Optimisation of the surface electrostatics as a strategy for cold adaptation of uracil-DNAN-glycosylase (UNG) from Atlantic cod.
Moe E., et. al. (2004)
Journal of Molecular Biology. 343(5): 1221-1230.
- Increased flexibility as a strategy for cold adaptation.
Olufsen M., et al (2005)
The Journal of Biological Chemistry. 280: 18042-18048.
- 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 A.O. (2007)
Journal of Molecular Graphics and Modelling. 26(1): 124-134.
Papers showing use of Uracil-DNA glycosylase
- Hypermethylated DNA, a circulating biomarker for colorectal cancer detection
Rasmussen SL, Krarup HB, Sunesen KG, Johansen MB, Stender MT, et al.
PLOS ONE 12(7), 2017. doi.org/10.1371/journal.pone.0180809
- Biofilm formation and antibiotic resistance in Klebsiella pneumoniae urinary strains
Vuotto, C., Longo, F., Pascolini, C., Donelli, G., Balice, M.P., Libori, M.F., Tiracchia, V., Salvia, A. and Varaldo, P.E.
J Appl Microbiol (2017), 123: 1003–1018. doi:10.1111/jam.13533
- Cell-free DNA promoter hypermethylation in plasma as a diagnostic marker for pancreatic adenocarcinoma
Henriksen SD, Madsen PH, Larsen AC, et al. (2016)
Clinical Epigenetics, 8, 117. http://doi.org/10.1186/s13148-016-0286-2
- Inter- and intraspecific variation in the surface pattern of the dermal bones of two sturgeon species
Thieren, E., Ottoni, C., Popović, D. and Van Neer, W. (2016)
Journal of Applied Ichthyology. doi: 10.1111/jai.13091
- A Concentrated Hydrochloric Acid-based Method for Complete Recovery of DNA from Bone
Leon Huynen Ph.D., David M. Lambert Ph.D.
J Forensic Sci, 60: 1553–1557. doi:10.1111/1556-4029.12846
- A New High-Throughput Approach to Genotype Ancient Human Gastrointestinal Parasites
Nathalie M. L. Côté, Julien Daligault, Mélanie Pruvost, E. Andrew Bennett, Olivier Gorgé, Silvia Guimaraes, Nicolas Capelli, Matthieu Le Bailly, Eva-Maria Geigl and Thierry Grange
PLoS One. 2016; 11(1): e0146230.
- Complex Species Status for Extinct Moa (Aves: Dinornithiformes) from the Genus Euryapteryx
Leon Huynen and David M. Lambert* (2014)
PLoS One. 2014; 9(3): e90212. doi: 10.1371/journal.pone.0090212
- Highly Informative Ancient DNA ‘Snippets’ for New Zealand Moa
Jonathan McCallum,1 Samantha Hall,1 Iman Lissone,2 Jennifer Anderson,2 Leon Huynen,1 and David M. Lambert1,* (2013)
PLoS One. 2013; 8(1): e50732. doi: 10.1371/journal.pone.0050732.
- Removal of deaminated cytosines and detection of in vivo methylation in ancient DNA.
Briggs A.W., et al. (2009)
Nucleic Acids Research. doi:10.1093/nar/gkp1163.
- Transcripts of developmentally regulated Plasmodium falciparum genes quantified by real-time RT-PCR.
Blair P.L., et al. (2002)
Nucleic Acids Research. 30(10): 2224-2231.
- An Efficient Multistrategy DNA Decontamination Procedure of PCR Reagents for Hypersensitive PCR Applications.
Champlot Sophie, Camille Berthelot, Mélanie Pruvost, E. Andrew Bennett, Thierry Grange, Eva-Maria Geigl. (2010)
PLoS ONE 5(9): e13042. doi:10.1371/journal.pone.0013042.
- Development of a novel rapid assay to assess the fidelity of DNA double-strand-break repair in human tumour cells.
Collis S.J., et al. (2002)
Nucleic Acids Research. 30(2): e1.
- Mutational analysis of the engrailed homeodomain recognition helix by phage display.
Connolly J., et al. (1999)
Nucleic Acids Research. 27(4): 1182-1189.
- A novel method employing UNG to avoid carry-over contamination in RNA- PCR.
Epstein U.J., et al. (1993)
Nucleic Acids Research. 21(16): 3917-3918.
- Avoiding false positives with PCR.
Kwok S., Higuchi R. (1989)
Nature. 339: 237 – 238.
- Direct isolation of poly(A)+ RNA from 4 M guanidine thiocyanate-lysed cell extracts using locked nucleic acid-oligo(T) capture.
Jacobsen N., et al. (2004)
Nucleic Acids Research. 32(7): e64.
- Quantitative assessment of the effect of uracil-DNA glycosylase on amplicon DNA degradation and RNA amplification in reverse transcription-PCR.
Kleiboeker S.B. (2005)
Virology Journal. 2: 29.
- Uracil-DNA glycosylase (UNG) influences the melting temperature (T(m)) of herpes simplex virus (HSV) hybridization probes.
Leblanc J.J., Pettipas J., Campbell S.J., Davidson R.J., Hatchette T.F. (2008)
J Virol Methods. 151(1): 158-60. Epub May, 12. 2008.
- Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions
Longo M.C., et al. (1990)
Gene. 93: 125-128.
- Minimizing DNA contamination by using UNG-coupled quantitative real-time PCR on degraded DNA samples: application to ancient DNA studies.
Pruvost M. et al. (2005)
BioTechniques. 38: 569-575.
- Determination of detection and quantification limits for SNP allele frequency estimation in DNA pools using real time PCR.
Schwarz G., et al. (2004)
Nucleic Acids Research. 32(3): e24-.
- Relationships between yeast Rad27 and Apn1 in response to apurinic/apyrimidinic (AP) sites in DNA
Wu X., Wang Z. (1999)
Nucleic Acids Research. 27(4): 956-962.
- A novel real-time PCR assay for quantitative analysis of methylated alleles (QAMA): analysis of the retinoblastoma locus.
Zeschnigk M., et al. (2004)
Nucleic Acids Research. 32(16): e125-.
- Uracil-DNA glycosylase-treated reverse transcription loop-mediated isothermal amplification for rapid detection of avian influenza virus preventing carry-over contamination
Kim, E. M.; Jeon, H. S.; Kim, J. J.; Shin, Y. K.; Lee, Y. J.; Yeo, S. G.; Park, C. K. (2016)
J Vet Sci 2016, 17 (3), 421-5.