Download Directions for Use Uracil-DNA Glycosylase (UNG), Cod

Directions for Use
Uracil-DNA Glycosylase (UNG), Cod
Code
Description
Size
1B1634-1KU
Uracil DNA Glycosylase (UNG), Cod
1000units (1 Unit/µL)
1B1634-0.1KU
Uracil DNA Glycosylase (UNG), Cod
100 units (1 Unit/µL)
General Information
Uracil-DNA Glycosylase (UNG), Cod is a thermolabile recombinant enzyme produced in E. coli
(ung-) by a modified ung gene derived from Atlantic Cod. It degrades uracil-containing singleand double-stranded DNA, but not RNA or thymidine-containing DNA, by hydrolyzing the Nglycosidic bond between deoxyribose sugar and the base in uracil. This generates alkalinesensitive apyramidinic sites in the DNA that will be cleaved upon a combination of alkaline
conditions and high temperature.
Pretreatment of samples with UNG prevents PCR carryover contamination in labs that
substitute dUTP in place of dTTP during all amplification reactions. PCR products containing
uracil become substrates for UNG and will be degraded if they are present in subsequent
reaction mixtures subjected to UNG treatment. Only DNA templates containing thymidine are
not degraded by the treatment and will be amplified.
Recombinant cod UNG is irreversibly inactivated upon heating, which enables long-term storage
and subsequent analysis of post-PCR amplicons in applications such as cloning and
sequencing. UNG is compatible with PCR, qPCR and one-step qRT-PCR and works in all
commercially available master mixes. All amplification reactions must use dUTP-containing
dNTP mixtures in order for the UNG decontamination method to be effective.
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Complete, irreversible heat-inactivation
Prevents carryover contamination in PCR, qPCR and qRT-PCR
Effective with only a 5 minute incubation step
Storage/Stability
Product is stable at least 2 years when stored frozen (-20 – 0°C). Product may be stored cold
(4 – 8°C) for up to 6 months.
Product Use Limitations
For research use only. Not for therapeutic or diagnostic use.
AMRESCO, LLC Corporate Headquarters, 28600 Fountain Parkway, Solon, OH 44139
Directions for Use
Protocol/Procedure:
Contamination Control in PCR and qPCR
Note: Ensure that dUTP is used instead of dTTP for PCR reactions.
The UNG storage buffer contains 20 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM DTT, 0.1% (w/v)
Triton-X-100 and 50% (w/v) Glycerol.
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Add 0.25 U of UNG directly to a 25 µL PCR Reaction.
Incubate the reaction 5 minutes at room temperature (20 – 25°C).
Perform the PCR amplification.
PCR product may be stored at -20°C or 4°C indefinitely until required for subsequent
analysis.
Contamination Control in One-step qRT-PCR
Note: The working temperature range for AMRESCO’s UNG is 20 – 40°C, with inactivation of
activity at 45°C and complete, irreversible inactivation at 55°C. Therefore, UNG is compatible
with one-step reverse transcriptase qPCR reactions, since it will not actively degrade the uracil
that is being incorporated into the cDNA during reverse transcription.
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Add 0.25 U of UNG directly to a 25 µL qRT-PCR reaction.
Incubate the reaction 5 minutes at room temperature (20 – 25°C).
Reverse transcribe RNA at 50 – 55°C.
Perform the PCR amplification.
qRT-PCR product may be stored at -20°C or 4°C indefinitely until required for subsequent
analysis.
Frequently Asked Questions
What are the advantages of AMRESCO’s Uracil-DNA Glycosylase (UNG), Cod?
 With complete, irreversible inactivation, AMRESCO’s UNG will not degrade new dUTPcontaining PCR amplicons, so that they may be used in cloning, sequencing, restriction
digestion, etc.
 AMRESCO’s UNG is inactive at temperatures ≥ 45°C, which enables pre-treatment of
one-step reverse transcription reactions with the enzyme prior to the cDNA synthesis
step. Unlike other Uracil-DNA Glycosylases, AMRESCO’s recombinant cod UNG will
not degrade the cDNA, which will have incorporated dUTP. It will only degrade
contaminating PCR amplicons in the pretreatment step.
 The enzyme is active at room temperature and can prevent carryover contamination in
amplification reactions with a fast 5 minute pretreatment.
AMRESCO, LLC Corporate Headquarters, 28600 Fountain Parkway, Solon, OH 44139
Directions for Use
What is the source of AMRESCO’s UNG?
 The recombinant cod UNG is produced in an ung negative E. coli strain and is designed
to replace the native UNG enzyme originating in Atlantic cod (Gadus morhua).
What is the stability of recombinant cod UNG?
 UNG can tolerate multiple freeze-thaw cycles. It may also be stored cold (2 – 8°C) for
up to 6 months.
How should UNG be heat inactivated?
 This enzyme is completely and irreversibly inactivated by incubation for 20 minutes at
55°C or at 95°C for 1 second.
How is a Unit of Cod UNG defined?
 One Unit will release 1 nmol uracil from uracil-containing DNA per hour at 37°C.
Can AMRESCO’s UNG replace E. coli UDG in all applications?
 AMRESCO’s UNG can replace E. coli UDG in all applications except those that require
activity above 40°C.
Why must dUTP be used in place of dTTP in every amplification reaction?
 dUTP-containing DNA will be degraded by UNG, but dTTP- containing DNA will not. If
amplicons with dTTP are present in the lab, they may potentially contaminate future
reactions because they will not be degraded by UNG treatment.
What is the advantage of a completely, irreversibly heat-inactivated UNG?
 UNG (or UDG) enzymes from other sources, which are not irreversibly inactivated,
eventually reactivate and degrade the new dUTP-containing amplicons even without the
deliberate addition of more UNG. AMRESCO’s UNG is irreversibly heat-inactivated,
ensuring that the amplicon may be stored or used in further applications without being
degraded. The sample will remain intact unless it is deliberately treated with UNG.
What are the buffer and temperature requirements for UNG activity?
 UNG is active in all common PCR, qPCR and qRT-PCR buffers and master mixes. Its
activity in common Taq buffers with 1.5 – 4 mM MgCl2 is close to 100%. The optimal
salt concentration is 50 mM NaCl, while excess salt decreases enzyme activity. UNG is
active between pH 7 – 9, but is optimal at pH 7.5. Effective temperatures range from 20
– 40°C, with optimal activity at 37°C.
How much UNG should be added per reaction?
 Typically, 0.1 – 1 U per 50 µL reaction works well, but each application is different and
the amount should be determined empirically.
What is the molecular weight of AMRESCO’s recombinant cod UNG?
 UNG is a 28 kDa protein.
AMRESCO, LLC Corporate Headquarters, 28600 Fountain Parkway, Solon, OH 44139
Directions for Use
Does Uracil Glycosylase Inhibitor (UGI) inhibit UNG?
 AMRESCO’s UNG does not require inhibitors due to its irreversible inactivation at 55°C.
However, it is does bind UGI in a 1:1 ratio and will be inhibited by it.
Will UNG remove uracil from both ss- and dsDNA with the same efficiency?
 No, UNG removes uracil from ss-DNA at a rate of 200% compared to dsDNA.
Will UNG remove uracil from RNA? Will it work with short oligos (e.g. 25-mer)?
 UNG does not act on uracil on the ribose sugar backbone. It will work on uracilcontaining oligos as small as 5-mers.
What is the difference between UNG and UDG?
 The E. coli enzyme Uracil DNA Glycosylase, which may also be called Uracil-NGlycosylase, is abbreviated frequently as UDG or UNG. Both names and abbreviations
refer to the protein encoded by the ung gene and are correct. Although VWR Life
Science AMRESCO’s recombinant UNG is derived from Atlantic cod, the E. coli enzyme
was the first to be identified.
Does UNG eliminate contamination 100% in PCR?
 UNG does degrade all uracil-containing DNA from prior PCR reactions, but may be less
efficient at decontaminating very small amplicons containing few uracils. Standard
precautions for preventing PCR contamination should still be in use, as new reactions
may be contaminated at any time by DNA lacking uracil.
How should PCR reactions treated with UNG be stored after amplification?
 UNG is irreversibly heat inactivated before the completion of PCR amplification, enabling
the long-term storage of new uracil-containing PCR products without the need for
product purification or concern about degradation. The PCR product may be stored
without time or temperature limitations and may be used directly in downstream
applications, such as cloning or sequencing.
Can uracil-containing DNA be used for restriction digests and molecular cloning?
 Yes, uracil-containing DNA may work in restriction digestion if the required restriction
enzyme is capable of digesting dUTP-containing DNA. For example, EcoRI and BamHI
will work well with dUTP-containing DNA, whereas HindII and HindIII show reduced
activity. Cloning of dUTP-containing DNA works as well as it does for dTTP-containing
DNA in cloning, but should be done using ung- bacterial hosts.
Can uracil-containing DNA be used in analysis of protein-DNA interactions?
 dUTP-containing DNA may interfere with protein binding or DNA-protein interaction
studies.
Can uracil-containing DNA be used for sequencing?
 dUTP-containing DNA performs as well as dTTP-containing DNA in dideoxy sequencing.
AMRESCO, LLC Corporate Headquarters, 28600 Fountain Parkway, Solon, OH 44139
Directions for Use
Can uracil-containing DNA be used as a probe for hybridization?
 Yes, it will perform as well as DNA with dTTP in hybridization.
Does uracil-containing DNA migrate normally in electrophoresis and can it be visualized
with DNA stains?
 Yes, it will migrate on gels in the same way and will be stained with the same efficiency
as dTTP-containing DNA.
How can there be a DNA band in a negative control even when the PCR reactions were all
treated with UNG?
 Although UNG will degrade all uracil-containing DNA, it is still possible to have
contamination from non-substrate DNA containing thymidine. This contamination may
be introduced from pipettes, contaminated reagents, etc. Always use standard
precautions for preventing contamination.
References:
1. Purification and characterization of a cold-adapted uracil-DNA glycosylase from Atlantic cod
(Gadus morhua). Lanes O., et al. (2000) Comparative Biochemistry and Physiology – Part
B: Biochemistry & Molecular Biology. 127: 399-410.
2. 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.
3. Transcripts of developmentally regulated Plasmodium falciparum genes quantified by realtime RT-PCR. Blair P.L., et al. (2002) Nucleic Acids Research. 30(10): 2224-2231.
4. 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.
5. 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.
6. Mutational analysis of the engrailed homeodomain recognition helix by phage display.
Connolly J., et al. (1999) Nucleic Acids Research. 27(4): 1182-1189.
7. 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.
8. Avoiding false positives with PCR. Kwok S., Higuchi R. (1989) Nature. 339: 237 – 238.
AMRESCO, LLC Corporate Headquarters, 28600 Fountain Parkway, Solon, OH 44139
Directions for Use
9. 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.
For Technical Support
Toll Free: 1-800-610-2789 (USA & Canada)
Fax: (440) 349-0235
Email: [email protected]
AMRESCO, LLC
A VWR Company
Corporate Headquarters
28600 Fountain Parkway
Solon, Ohio USA 44139-4300
Tel: 440/349-1199
Fax: 440/349-1182
www.amresco-inc.com
Uracil-DNA Glycosylase (UNG), Cod
ZY0615
Rev. 1 12/2015
© Copyright 2010 by AMRESCO, LLC
All Rights Reserved.
AMRESCO® is a registered trademark of AMRESCO, LLC
AMRESCO, LLC Corporate Headquarters, 28600 Fountain Parkway, Solon, OH 44139