UNG Heat Labile
Description
Definition and Biochemical Properties
UNG Heat Labile (Uracil-DNA Glycosylase) is a recombinant enzyme that hydrolyzes the N-glycosidic bond between uracil and deoxyribose in DNA, creating abasic sites susceptible to strand breakage under heat or alkaline conditions . Key characteristics include:
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Thermolability: Rapidly inactivated at temperatures >50°C (half-life of 2 minutes at 40°C in dilute buffers) .
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Activity: Degrades single- and double-stranded uracil-containing DNA but is inactive on RNA or uracil-free DNA .
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Storage: Typically supplied in 20–50% glycerol buffers at -20°C .
Mechanism of Action
UNG Heat Labile prevents PCR carryover contamination through a two-step process:
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Pre-PCR treatment: Incubation at 20–25°C cleaves uracil bases from contaminating DNA amplified in prior reactions (e.g., dUTP-containing amplicons) .
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Heat inactivation: During PCR initialization (95°C), the enzyme is irreversibly denatured, preventing interference with new amplification .
This mechanism ensures that only target DNA (lacking uracil) is amplified, while contaminants are degraded .
Key Uses
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PCR carryover prevention: Integrated into master mixes for diagnostic RT-PCR and qPCR .
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One-step RT-PCR compatibility: Thermolability allows seamless use without post-amplification DNA damage .
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High-sensitivity assays: Critical for pathogen detection (e.g., Porcine arterivirus) to avoid false positives .
Performance Data
Research Findings and Limitations
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Temperature sensitivity: UNG activity at 25°C increases C<sub>T</sub> values by 0.53–2.13 cycles in RT-PCR, reducing false positives but marginally lowering sensitivity .
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Concentration dependence: Higher UNG doses (1 U vs. 0.5 U) exacerbate C<sub>T</sub> shifts, necessitating optimization .
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Residual activity risks: Non-Cod UNG variants may reactivate during prolonged storage, damaging dUTP-containing amplicons .
Formulation and Stability
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Buffer composition: 20 mM Tris-HCl, 100 mM KCl, 0.1–1 mM EDTA, 50% glycerol .
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Storage: Stable at -20°C for >1 year; avoid freeze-thaw cycles .
Best Practices for Use
Product Specs
Uracil-DNA glycosylase (UNG), also known as Uracil-N-glycosylase, is an enzyme responsible for removing uracil from DNA. Uracil can occur in DNA through two primary pathways: the spontaneous deamination of cytosine to uracil and the misincorporation of dUTP instead of dTTP during DNA synthesis. The presence of uracil in DNA can be detrimental as it can lead to mutations if not repaired. E. coli UNG is a valuable tool in molecular biology for various applications. It plays a crucial role in preventing PCR carryover contamination, ensuring accurate results in PCR experiments. UNG removes uracil residues from DNA, preventing the amplification of undesirable templates and enhancing the specificity of PCR amplification.
UNG, derived from a psychrophilic marine bacterium, is produced recombinantly in E. coli. It exists as a single, non-glycosylated polypeptide chain, meticulously purified using proprietary chromatographic techniques.
The UNG protein solution is supplied at a concentration of 1 U/µl in a buffer consisting of 20 mM Tris-HCl (pH 8.0 at 25°C), 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5% NP-40, 0.5% Tween-20, and 50% glycerol.
One unit of UNG activity is defined as the amount of enzyme required to release 1 nanomole of uracil from a uracil-containing DNA strand per hour at 37°C. This activity is measured under standard assay conditions, which include a reaction mixture containing 70 mM Tris-HCl (pH 7.5), 10 mM NaCl, 1 mM EDTA, and 0.1 mg/ml BSA.
The specific activity of this UNG enzyme is greater than or equal to 200,000 units per milligram of protein (≥ 200,000 U/mg).
The purity of the UNG enzyme is determined by SDS-PAGE analysis and is greater than 97.0%.
Treatment of 0.1 µg of uracil-containing DNA with 1 unit of UNG for 10 minutes at 37°C renders the DNA unsuitable for copying by DNA polymerase. While the enzyme can be 95% heat-inactivated by incubation at 95°C for 10 minutes, it is important to note that UNG may retain partial activity after this treatment. To prevent potential degradation of product DNA due to residual UNG activity, adding a uracil glycosylase inhibitor is recommended. As an alternative, reaction products can be immediately extracted using a phenol/chloroform extraction method.
Product Science Overview
Uracil DNA glycosylase (UDG), also known as uracil-N glycosylase (UNG), is an enzyme that plays a crucial role in the base excision repair pathway by removing uracil from DNA. Uracil can arise in DNA through the deamination of cytosine or the misincorporation of dUMP during DNA synthesis. The removal of uracil is essential to prevent mutations and maintain genomic stability.
Psychrophilic bacteria are microorganisms that thrive in extremely cold environments, such as the deep sea or polar regions. These bacteria have adapted to function optimally at low temperatures, making them a valuable source of cold-active enzymes. One such enzyme is the heat-labile uracil DNA glycosylase from a psychrophilic marine bacterium.
The heat-labile uracil DNA glycosylase from a psychrophilic marine bacterium, specifically strain BMTU3346, has been extensively studied. This enzyme is characterized by its cold-active nature, with an optimal temperature of 35°C and a half-life of only 2 minutes at 40°C . The gene encoding this enzyme has been cloned, sequenced, and expressed in Escherichia coli, allowing for detailed studies of its properties and potential applications .
The gene encoding the heat-labile UDG from strain BMTU3346 was successfully cloned and sequenced. The amino acid sequence of this enzyme shows significant identity (39.1%–46.2%) to UDGs from mesophilic bacteria . However, the primary structure of the heat-labile UDG revealed 22 differences compared to the consensus sequence derived from bacterial UDGs . These differences are believed to contribute to the enzyme’s thermolability, although no dominant features such as extended surface loops or a decrease in arginine or proline residues were observed .
The heat-labile UDG from the psychrophilic marine bacterium exhibits high selectivity for the excision of uracil from DNA. It hydrolyzes the N-glycosidic bond linking uracil to the deoxyribose sugar, generating an abasic site. This site is subsequently processed by an apurinic/apyrimidinic (AP)-endonuclease and a phosphodiesterase, followed by gap filling by a DNA polymerase and sealing by a DNA ligase . The enzyme shows no activity against normal DNA bases or uracil in RNA .
The unique properties of the heat-labile UDG make it a valuable tool in molecular biology. Its cold-active nature and rapid inactivation at higher temperatures allow for precise control in various applications, such as polymerase chain reaction (PCR) protocols. The enzyme can be used to remove uracil from DNA templates, preventing carryover contamination in PCR reactions .
