UNG Vs UDG

What is UNG/UDG?
Perhaps you've wondered why some master mixes contain UNG and other master
mixes contain UDG and thought to yourself, what is the difference?
To answer this question, we first need to establish that for qPCR practical purposes
there is no difference. But, it is a little more complicated than just stating there is no
difference. Uracil-DNA glycosylases (UDGs) are evolutionary, well-preserved DNA-
repair enzymes. The term UDG refers to a superfamily of enzymes comprising six
sub-families. Family I UDG enzymes are called UNG, after the uracil-N-glycosylase
gene [6]. The terms UDG and UNG are commonly used interchangeably because they
perform the same function in qPCR—namely to prevent carryover contamination.
The biological function is to remove uracil—normally found in RNA—from DNA,

creating free uracil and alkali-sensitive apyrimidic sites in DNA [2,6]. UNG removes
uracil incorporated into single- and double-stranded DNA by catalyzing hydrolysis in
the N-glycosylic bond between uracil and sugar [7], 2008). Most notably, the enzyme
prefers to act on single-stranded uracil templates [6].
How is UNG/UDG used in qPCR?

As PCR can amplify such tiny amounts of DNA, preventing contamination is
essential: even small amounts of contamination can produce false positives in your
experiments. Contamination can include cross-contamination from other samples,
DNA contamination from elsewhere in the laboratory, and carryover contamination
from amplification products and primers used in prior PCR experiments [4]. The latter
causes many of the false positive results seen in PCR [3]. Preventing contamination
is difficult. Special laboratory procedures must be in place to ensure leftover DNA
residue is not re-amplified in subsequent experiments.
UNG can specifically degrade products that have already been through the PCR
process. UNG allows previous PCR amplifications or mis-primed, nonspecific
products to degrade, leaving native nucleic acid templates intended for amplification
intact. UNG activation occurs as the first step of PCR at a 50°C incubation for 2
minutes.
Does UNG affect other aspects of qPCR?

UNG is active on single- and double-stranded dU-containing DNA, but dUTP is not a
substrate for UNG. Taq polymerase and other components of the PCR mixture are not
affected by UNG treatment. Only carryover product will be removed with UNG
treatment [8].
The dU-containing PCR product behaves like native dT-containing DNA in blotting,
cloning and sequencing, and the presence of UNG will not affect most post-PCR
analyses. In addition, UNG does not affect the electrophoretic mobility or ethidium–
bromide-staining efficiency of DNA, and will not affect your experimental results.
Page 1 of 3
What is UNG/UDG?
To prevent carryover contamination in your qPCR, use a master mix that contains
either UNG or UDG.
Troubleshooting and when not to use UNG

Despite the advantages that UNG offers, E. coli UNG is not fully heat-deactivated and
can degrade PCR products over time, which will affect the results of your PCR
experiments. If you are running a genotyping experiment and plan to perform an end-
point read at a later date, it is advisable to not use UNG.
E. coli UNG is also not recommended for 1-step RT-PCR application as the reverse
transcription step to create cDNA would incorporate dU-nucleotides thus degraded
by UNG in the reaction. The remedy is to convert RNA to cDNA in separate reactions.
Alternatively, there are 1-step RT-PCR master mix that uses dU-nucleotides but uses
a heat-labile UNG (cloned from Atlantic cod species). The heat labile UNG can be in-
activated during the 50-55°C reverse transcription step thus the first strand cDNA
that contains dU bases will not be degraded.
Although UNG actively prevents future contamination of samples, it cannot remove

preexisting contamination from standard dTTP-containing PCR products. However,
good laboratory practices can solve this issue.
The sequence being amplified should contain dA and dT nucleotides. Only DNA
sequences with these nucleotides will lead to dU-containing PCR products that can
be degraded by UNG.
Primers should contain dA-nucleotides near their 3' ends, so that the primer-dimers
generated are degraded by UNG at least as efficiently as dU-containing PCR
products. The further a dA-nucleotide is from the 3' end, the more likely the partially
degraded primer-dimers may serve as templates for a subsequent PCR amplification.
Producing such primer-dimers could compromise the amplification of the desired
target region. If primers with dA-nucleotides near the end cannot be used, consider
primers with 3' terminal dU-nucleotides. Terminal dU-nucleotides are not substrates
for UNG; these primers will not be degraded [1].
UNG is also not suitable for use in amplifying dU-containing PCR products, as in
nested-PCR protocols, as the enzyme will degrade the dU-containing PCR product,
preventing further amplification [5]. Furthermore, UNG is not suitable for amplifying
bisulfite converted DNA template. This is because bisulfite converts unmethylated
cytosine bases into uracil residues.
In general, any time you want to work with the amplicon after the run is over, but
perhaps not immediately, it would be best to use a master mix without UNG.
Page 2 of 3
What is UNG/UDG?
As UNG has activity below 55°C, this should be the minimum annealing temperature
for PCR amplification, to avoid degradation of newly synthesized dU-containing PCR
products by residual UNG activity, even after a long initial denaturation step [8].
Page 3 of 3

UNG Vs UDG

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UNG Vs UDG

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What is UNG/UDG?

The biological function is to remove uracil—normally found in RNA—from DNA,

How is UNG/UDG used in qPCR?

Does UNG affect other aspects of qPCR?

Troubleshooting and when not to use UNG

Although UNG actively prevents future contamination of samples, it cannot remove

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