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Cat. No.: UDG-1k (for 1000U)

Cat. No.: UDG-5k (for 5000U)

For the heat-labile version, please visit HL-UDG.

Description

Uracil-DNA Glycosylase (E. coli), also known as E. coli Uracil-DNA Glycosylase (UDG) or E. coli Uracil N-Glycosylase (UNG), is derived from Escherichia coli (E. coli). It catalyzes the hydrolysis of the N-glycosidic bond between uracil (dU) bases and deoxyribose in uracil-containing DNA strands, thereby releasing free uracil. Uracil-DNA Glycosylase (UDG) can hydrolyze uracil from both single-stranded and double-stranded DNA containing dU, but it does not act on RNA or DNA oligomers shorter than 6 bases containing dU.

UDG is primarily used to eliminate contamination from PCR amplification products. The principle behind its use for contamination prevention is as follows: In PCR reactions, dUTP is added to replace dTTP, incorporating dU into the DNA to form uracil-containing PCR products. In subsequent PCR reactions, UDG selectively cleaves single-stranded or double-stranded DNA containing dU that may have been introduced as contamination, thus preventing any negative impact from previous PCR amplification products on the current reaction.

Activity Definition

One unit of enzyme activity is defined as the amount that catalyzes the release of 60 pmol of uracil per minute from double-stranded, uracil-containing DNA. Activity is measured by the release of [3H]-uracil in a 50 µl reaction containing 0.2 µg DNA (104–105 cpm/µg) in 30 minutes at 37℃.

Source

Recombinant, expressed, and purified from Escherichia coli (E. coli).

Purity

Free of endo- or exonucleases, RNase, and phosphatase activities other than UDG enzyme activity.

Applications

• Preventing cross-contamination of PCR products.
• Single nucleotide polymorphism detection (GMPD).
• Site-specific mutagenesis.
• Studying protein-DNA interactions.
• SNP genotyping.
• Cloning of PCR products.
• Preparing PCR products or cDNA with single-stranded overhangs.

Inactivation or Inhibition

Heating at 95℃ for 10 minutes can inactivate 95% of E. coli UDG. Since partial activity may remain after 10 minutes at 95℃, it is recommended to add UDG inhibitors (such as Ugi protein from Bacillus subtilis bacteriophage PBS2 or p56 protein from phi29 bacteriophage) to further inhibit its enzyme activity and prevent continued degradation of dU-containing PCR products.

Storage

Store at -20℃, effective for at least one year.

Precautions

• E. coli UDG is active in most PCR reaction buffers; however, it is recommended to test compatibility with your specific PCR or RT-PCR system upon first use.
• DNA strands containing abasic sites generated by E. coli UDG digestion can be removed by heat, alkaline treatment, or nuclease treatment. The heating steps in the PCR process usually ensure complete cleavage of UDG-digested sites.
• E. coli UDG is active over a broad pH range, with an optimal pH of 8.0. It does not require divalent cations for activity and is inhibited by high ionic strength (> 200 mM).
• E. coli UDG can eliminate unintended dUTP-containing PCR products before the reaction, preventing false positives due to contamination.
• E. coli UDG may exhibit residual activity at lower temperatures after heat denaturation due to refolding. Therefore, it is recommended to use an annealing temperature of 55℃ or higher in subsequent PCR steps.
• While E. coli UDG can be used in conventional PCR or qPCR amplification systems for DNA or cDNA, it is generally not recommended for RT-PCR systems. Under reverse transcription conditions, E. coli UDG remains active and may digest newly synthesized cDNA.
• If E. coli UDG is to be used in RT-PCR systems, it is necessary to separate the reverse transcription and PCR steps, avoiding dUTP during reverse transcription. Post-reverse transcription, treat with E. coli UDG before proceeding with conventional PCR or qPCR. Alternatively, adding UDG inhibitors (such as Ugi protein from Bacillus subtilis bacteriophage PBS2 or p56 protein from phi29 bacteriophage) can further inhibit E. coli UDG activity.
• This product is intended for use by trained professionals for scientific research purposes only. It is not to be used for clinical diagnosis or treatment, not for use in food or pharmaceuticals, and should not be stored in residential settings.

Related:

• Uracil N-Glycosylase
• Heat-Labile Uracil DNA Glycosylase (HL-UDG)
• Heat-Labile Uracil DNA Glycosylase 2.0 (HL-UDG 2.0)
• Thermostable UDG (Uracil-DNA Glycosylase)

Only for research and not intended for treatment of humans or animals

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