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YEASEN UDG:The "anti-fouling expert" in PCR reactions

The most frequent source of false-positive PCR findings is aerosol contamination in the operational environment. UDG enzyme (Uracil DNA Glycosylase) was found by American scientist Lindahl in Escherichia coli and Bacillus subtilis. The UDG enzyme and dUTP may be utilized to construct a PCR anti-pollution system that ensures accurate PCR amplification results. So what are UDG enzymes? What is its mechanism of action?

 

1. UDG enzyme's mechanism
2. Variations of UDG enzymes
3. YEASEN UDG
4. FAQ
5. Order Guide

 

1. UDG enzyme's mechanism

PCR is an extremely sensitive amplification technique and is susceptible to contamination. Small amounts of foreign DNA contamination can be amplified along with the template of interest. Therefore, all PCR reagents used for clinical tests must have UDG enzyme technology to prevent contamination. The PCR amplification fragments used for clinical detection are usually about 300bp, but the decontamination effect of UV irradiation is not effective for fragments below 500bp. Therefore, the preventive effect of UDG has been paid more and more attention. UDG is a monomeric protein with relatively stable physicochemical properties and a small molecular weight of 25KDa. It is widely present in various prokaryotic and eukaryotic organisms.
UDG is inactive against RNA, but can catalyze single- and double-stranded DNA containing uracil and then release free uracil. Therefore, the UDG enzyme is mainly used for the anti-contamination of PCR amplification products. Its working principle is: that UDG enzymes can catalyze the hydrolysis of the N-glycosidic link between the uracil base and the sugar-phosphate backbone in ssDNA and dsDNA, but they are inactive to RNA. When utilizing the UDG enzyme, dUTP is substituted for dTTP in PCR amplification. UDG enzyme can digest uracil-containing PCR products and remove leftover PCR contamination before the initiation of the PCR process.
And UDG catalyzes the hydrolysis of N-nucleoside bonds, which can also lead to the recognition of abasic sites by endonuclease IV and exonuclease III. And cut the abasic site at the 5' end to generate 3' hydroxyl and 5' phosphate deoxyribose. The other DNA repair enzymes (such as DNA polymerase, DNA ligase, etc.) will act further to complete the entire base excision repair. In addition to participating in the DNA repair process, UDG is also related to the body's immune function, participating in the production of immune molecule antibody diversity and the natural antiviral defense process in cells.
UDG has very strict substrate specificity. First of all, uracil is the only base recognized by the UDG enzyme, uracil must be a component of deoxynucleosides, and uracil must be in the polymer. Studies of substrate specificity have pointed out that the sugar-phosphate backbone is the key to UDG recognition in the primary structure of the substrate. The recognition and binding sites of UDG are structurally dual, and although the enzyme is relatively non-specific for polynucleotides, it can bind to single- and double-stranded DNA and RNA. But it only acts on the uracil residue on the 2'-deoxyribose sugar in the polynucleotide.
dUTP/UDG enzyme is utilized as an anti-pollution component of the PCR reaction system. During a period of 25
for 10 minutes before the PCR process, The UDG enzyme catalyzes the hydrolysis of the N-glycosidic link between the uracil base and the sugar-phosphate backbone in the dU-DNA chain to yield a pyrimidine-deficient site and free uracil. The UDG enzyme was then inactivated by heating it for 2 minutes at 95 . Moreover, the high temperature allows the phosphate backbone of the pyrimidine-deficient site to be readily hydrolyzed and broken, hence removing uracil contamination in PCR products.
Natural and unmodified DNA lacks dU, is unaffected by UDG enzymes, and can be utilized as a template for PCR/qPCR amplification.

Figure 1. Schematic diagram of the dUTP/UDG enzyme anti-pollution system to remove residual amplification contaminants

 

2. Variations of UDG enzymes

Two types of normal UDG enzymes developed from Escherichia coli and Bacillus subtilis and two types of heat-labile UDG enzymes generated from psychrophilic marine bacteria comprise the majority of commercially available UDG enzymes at present. Normal UDG enzymes are resistant to heat, and a minor amount of uracil-DNA glycosylase activity will continue after 10 minutes at 95 . It prevents the destruction of dU-containing amplification products at room temperature by residual activity that may occur following the inactivation of traditional UDG enzymes.
The normal bases A, T, G, and C constitute normal DNA. Through PCR reaction, a large amount of uracil-containing DNA (U-DNA) is amplified with dUTP instead of traditional dTTP. This is an efficient substrate for thermosensitive UDG. When the amplification product acts as a "residual contaminant", it can generate a purine pyrimidine sites through the hydrolysis of thermosensitive UDG. When PCR predenaturation is performed at this site, the DNA strand is broken at these sites, and the template function is lost.

 

3. YEASEN UDG

3.1  Product Features

Psychrophilic sea bacteria are the source of Uracil DNA Glycosylase (UDG/UNG), 1 U/μL (Cat#10707ES). It is an Escherichia coli-expressed and purified recombinant protein. It is active between 25 ℃ and 37, and 95℃ for 5-10 minutes causing irreversible inactivation.    

  • 10 U of this product was detected by E.coli 16S rDNA-specific TaqMan qPCR, and the result showed that E.coli genome residue was fewer than 10 copies. 
  • No nucleic acid endonuclease, exonuclease, and RNase residues.
  • Uracil is the sole base recognized by this enzyme.

3.2 How to use (for reference only)

Based on the demands of the experiment, the final concentration of dUTP may be varied between 0.2 mM and 0.6 mM, and 0.2 mM dTTP can be included selectively.

  • 1 U of UDG enzyme is added to the reaction system, which can digest 1 μg dU-DNA in 30 minutes.
  • Prior to the PCR procedure, 2 - 10 minutes of incubation at 25 ℃ to 37 ℃ was added to activate the UDG enzyme activity.
  • Subsequent PCR operations are performed based on the experimental needs.

3.3 0.025 U UDG enzyme can digest 360 ng of 200 bp dU-DNA completely

Electrophoresis results of 0.05 U, 0.025 U, 0.0125 U, 0.00625 U, 0.003125 U heat-labile UDG enzyme with 360 ng of 200 bp dU-DNA at 25 ℃ for 30 min (95 ℃, 2 min inactivation)

Figure 2. Electrophoresis results of 0.05 U, 0.025 U, 0.0125 U, 0.00625 U, 0.003125 U heat-labile UDG enzyme with 360 ng of 200 bp dU-DNA at 25 for 30 min.

3.4 Application Scenarios

  • Remove ssDNA and dsDNA, but they are inactive to RNA.
  • Eliminate false positive results due to contamination of PCR amplification products.
  • Compatible with PCR, qPCR, RT-qPCR, etc.

 

4. FAQ

Q1: Can UDG enzymes be used to already existed aerosolize amplification products?

A: Existing amplification product aerosol pollution does not include dU, thus UDG enzyme cannot operate. To avoid "future" aerosol pollution, choose alternate amplification sites, redesign primers and construct a dUTP/UDG enzyme anti-pollution system is suggested.

Q2: Will the amplified dU-DNA product from dUTP influence hybridization, sequencing, cloning, and digesting?

A: The dU-containing PCR products can be utilized for nucleic acid hybridization and sequencing routinely. In molecular cloning research, ligation products must be turned into UDG-deficient competent cells. EcoRI and BamHI are not impacted by dU in digestion experiments, but the efficiency of HindIII is reduced, and other endonuclease effects need to be tested.

Q3: The dU-containing PCR product was ligated and transformed, why no clone was found?

A: In molecular cloning, the ligation product must be converted into UDG-deficient competent cells for plasmid amplification.

Q4: Does UDG enzyme break RNA?

A: No. UDG enzymes serve a vital function in DNA repair in living organisms. It is capable of repairing the mutation of dC deamination to generate dU, hence preventing the conversion of GC base pairs to AT base pairs.

Q5: What is the buffer for the UDG enzyme's reaction?

A: UDG enzyme may function in several buffers, including regular Taq enzyme buffer, ligase buffer, and enzyme digestion buffer, among others.

Q6: Does the UDG enzyme have base-count requirements for DNA strands?

A: Uracil cannot be hydrolyzed from oligonucleotides with less than six bases.

 

5. Order Guide

The related products provided by Yeasen are as follow:

Table 1. Related products

Product Positioning

Product name

Cat#

heat-labile UDG

Uracil DNA Glycosylase (UDG/UNG), 1 U/μL

10303ES

dUTP

dUTP,100mM Solution (100 mM)

10128ES

dNTP

dNTP Mix (25 mM each)

10125ES

Reverse Transcriptase for RT-qPCR

Hifair ™ V Reverse Transcriptase (200 U/μL)

11300ES

Hotstart Taq DNA Polymerase for RT-qPCR

Hieff Unicon™ Hotstart Direct Taq DNA Polymerase (5 U/μL) (Inquire)

10717ES

Reverse Transcriptase for RT-Lamp

Hifair™ III Reverse Transcriptase (200 U/μL) (Inquire)

11111ES

Bst DNA Polymerase for RT-Lamp

Hieff™ Bst Plus DNA Polymerase (40 U/μL)

14402ES

RNase inhibitor

Murine RNase inhibitor (40 U/μl)

10603ES

Double-Block anti-Taq DNA Polymerase Antibody

Hieff™ Double-Block anti-Taq DNA Polymerase Antibody

31303ES

 

Regarding reading:

Murine RNase Inhibitors-Successfully eliminate RNase contamination and preserve RNA

 

References

[1] Li ZY, Liu MX. Application of Uracil DNA Glycosylase in PCR Anti-pollution[J]. China Medicine Guide, 2001, 3(4): 305-306.
[2] Nilsen H, Otterlei M, Haug T, et al. Nuclear and mitochondrial uracil-DNA glycosylases are generated by alternative splicing and transcription from different positions in the UNG gene[J ]. Nucleic Acids Res, 1997, 25 (4): 750-755.(IF 19.16)