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.

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.

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].

References

1. Delort AM, Duplaa AM, Molko D et al. (1985) Excision of uracil residues in DNA: Mechanism of action of Escherichia coli and Micrococcus luteus uracil-DNA glycosylases. Nucleic Acids Res 13:319–335. doi: 10.1093/nar/13.2.319.
2. Dianov G, Lindahl T (1994) Reconstitution of the DNA base excision-repair pathway. Curr Biol 4(12):1069–1076.
3. Kwok S, Higuchi R (1989) Avoiding false positives with PCR. Nature 339(6221):237–238. doi: 10.1038/339237a0.
4. Longo N, Berninger NS, Hartley JL (1990) Use of Uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene 93:125–128.
5. Seitz V, Schaper S, Droge A et al. (2015) A new method to prevent carry-over contaminations in two-step PCR NGS library preparations. Nucleic Acids Res 43(20):e135. doi: 10.1093/nar/gkv694.
6. Shormann N, Ricciardi R, Chattopadhyay D (2014) Uracil-DNA glycosylases—Structural and functional perspectives on an essential family of DNA repair enzymes. Protein Science 23(12): 1667–1685. doi: 10.1002/pro.2554.
7. Sidorenko VS, Mechetin GV, Nevinsky GA et al. (2008) Correlated cleavage of single- and double-stranded substrates by uracil-DNA glycosylase. FEBS Lett 582(3):410–404. doi: 10.1016/j.febslet.2008.01.002.
8. Tetzner R, Dietrich D, Distler J (2007) Control of carry-over contamination for PCR-based DNA methylation quantification using bisulfite treated DNA. Nucleic Acids Res 35(1):e4. doi: 10.1093/nar/gkl955.