Cod Uracil-DNA N-glycosylase (Cod UNG)
The ideal enzyme for use in PCR carry-over prevention
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Molecular structure of cod Uracil-DNA N-glycosylase (Leiros et al. 2003) |
Uracil-DNA N-glycosylase (UNG) from Atlantic cod is the first commercially available UNG enzyme that is completely and irreversibly inactivated by moderate heat treatment. The enzyme is produced in recombinant form from an E. coli ung- mutant that contains a modified cod UNG gene.
By PCR, a few molecules of target DNA may be amplified to detectable amounts. This makes the method very sensitive to contamination. The prime source of non-target contaminants is PCR products from previous PCR amplifications. When all PCR amplifications are made with dUTP instead of dTTP, PCR products become distinguishable from target DNA, and can be selectively degraded by UNG. A pre-incubation of the PCR reaction mix with UNG will remove Uracil from the DNA backbone, which leads to degradation of contaminant DNA during subsequent heating.
The problem with commercially available UNG enzymes is that they do not inactivate completely upon heat treatment and they may reactivate. Therefore special precautions must be made if the PCR products need to remain intact for further analysis. This is not a problem when Cod UNG is used, since this enzyme is completely and irreversibly inactivated after heat treatment.
Contamination control1) and genotyping
For contamination control in PCR-based genotyping or sequencing applications, Cod UNG is the only choice. All other UNG enzymes on the market will degrade the PCR product post-PCR, which may give poor and unreliable results. With our enzyme, contamination control is still possible even when the PCR product is to be sequenced or genotyped by single-nucleotide primer extension technology.
Cod UNG is therefore the natural choice for contamination control in strain genotyping kits for viruses or bacteria.
Particularly useful for contamination control1) in RT-PCR
When RNA is used as template for amplification (RT-PCR), effective contamination control may be difficult to achieve. This is because the cDNA generated by reverse transcriptase may get degraded by the presence of UNG. However, contamination control using UNG is still possible provided that the UNG enzyme is sufficiently heat labile. At 50 °C, a temperature commonly used for MMLV or AMV reverse transcriptases, Cod UNG is rapidly inactivated, giving minimal degradation of the generated cDNA. In addition, since Cod UNG is very efficient at low temperature, UNG preincubation can be done at a temperature low enough to minimize cDNA generation during this step.
These two properties combined makes contamination control feasible in RT-PCR with no loss of sensitivity.
Recombinant Cod UNG therefore facilitates contamination control also in qRT-PCR detection methods for RNA viruses like HIV, Hepatitis, or Avian Flu.
Availability:
Recombinant Cod UNG is available directly from Biotec Pharmacon and from USB Corp (Affymetrix, worldwide).
Specifications:
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. DNase, RNase or protease activities not detected.
Concentration: Minimum 1,000 Units/ml.
Properties:
Stability: Stable at -20 °C in storage buffer (20 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM DTT, 0.1 % Triton X-100, 50 % glycerol). Completely inactivated after 10 min at 60 °C.
Additional information on the properties of Cod UNG can be found in this pdf document
Literature:
Lanes,O., Guddal,P.H., Gjellesvik,D.R., Willassen,N.P. (2000). Purification and characterization of a cold-adapted uracil-DNA glycosylase from Atlantic cod (Gadus morhua). Comp Biochem Physiol B Biochem Mol Biol 127, 399-410.
Lanes, O., Leiros,I., Smalås, A.O., Willassen, N.P. (2002). Identification, cloning, and expression of uracil-DNA glycosylase from Atlantic cod (Gadus morhua): characterization and homology modeling of the cold-active catalytic domain. Extremophiles 6, 73-86.
Leiros, I., Lanes, O., Sundheim, O., Helland, R., Smalas, A.O., Willassen, N.P. (2001). Crystallization and preliminary X-ray diffraction analysis of a cold-adapted uracil-DNA glycosylase from Atlantic cod (Gadus morhua). Acta Cryst. D57, 1706-1708.
Leiros, I., Moe, E., Lanes, O., Smalas, A. O., and Willassen, N. P. (2003). The crystal structure of Uracil-DNA N-glycosylase from Atlantic cod (Gadus morhua) reveals cold-adapted features. Acta Cryst. D59, 1357-1365 . 2003.
Longo, M.C., Berninger, M.S., Hartley, J.L. (1990). Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene 93, 125-128.
Moe, E. The molecular basis for cold adaptation of Uracil-DNA N-glycosylase from Atlantic cod (Gadus morhua).2002. Thesis, University of Tromsø. 2002.
Kleiboeker, SB (2005). Quantitative assessment of the effect of uracil-DNA glycosylase on amplicon DNA degradation and RNA amplification in reverse transcription-PCR. Virology Journal 2, 29
1)The use of UNG in controlling contamination in PCR and related technologies may in some countries be subject to intellectual property rights, and may require a licence.
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