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| Product Name | Uracil-DNA Glycosylase (UDG, Heat-Labile) |
| Catalog No. | IEC-HMM-0058 |
| Description | A recombinant DNA repair enzyme that specifically hydrolyzes the N-glycosidic bond between uracil and the deoxyribose sugar in single- or double-stranded DNA containing uracil residues. The heat-labile variant is derived from a psychrophilic marine bacterium and is completely and irreversibly inactivated by brief heating at 50 °C, making it ideal for carryover prevention in PCR workflows. |
| Intended Use | For carryover contamination prevention in PCR and qPCR through the dUTP/uracil-DNA glycosylase system. Also used in site-directed mutagenesis, bisulfite conversion analysis, and enzymatic removal of uracil from DNA for downstream applications. |
| Principle / Technology | UDG scans DNA for uracil residues (arising from dUTP incorporation or cytosine deamination) and cleaves the uracil base from the sugar-phosphate backbone, generating abasic (AP) sites. In the dUTP carryover prevention system, PCR products synthesized with dUTP instead of dTTP serve as substrates for UDG, which degrades contaminating products before new amplification while leaving natural (thymine-containing) template DNA unaffected. |
| Detection Method | Activity measured by release of [³H]-uracil from poly(dA)·poly([³H]-dU) substrate. One unit releases 1 pmol uracil in 60 min at 37 °C. |
| Sample Type | PCR reaction mixes containing dUTP-substituted DNA. Also: DNA substrates from bisulfite treatment or synthetic oligonucleotides containing deoxyuridine. |
| Performance Range / Specifications | Active at 20-45 °C. Complete heat inactivation: 50 °C, 10 min. Compatible with standard PCR/qPCR buffer systems. |
| Sensitivity / LOD | Detects and cleaves uracil at single-base resolution in DNA substrates. |
| Specificity | Highly specific for uracil in DNA. Does not cleave thymine, cytosine, 5-methylcytosine, or RNA. Does not act on uracil in RNA. |
| Reaction Conditions / Protocol | Add 0.5-1 U UDG per 25 uL PCR reaction. Pre-incubate at 37 °C 10 min (carryover digestion). Heat inactivate: 50 °C 10 min (or combine with initial PCR denaturation at 95 °C, which also inactivates). Proceed with thermal cycling. |
| Components / Formulation | Heat-labile UDG (1 U/uL) in 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 50% glycerol. |
| Storage Conditions | Store at -20 °C to -30 °C. |
| Shelf Life | 24 months at -20 °C. |
| Package Specifications | 500 U (500 uL), 2,500 U (2.5 mL), 10,000 U (10 mL). |
| Product Form | Clear, colorless glycerol-based liquid. |
| Quality Control | Each lot tested for: uracil excision activity; absence of contaminating DNase, RNase, and non-specific endonuclease; functional carryover prevention in dUTP-based qPCR assay; complete heat inactivation at 50 °C verified by lack of activity after heating. |
| Key Features | Complete irreversible inactivation at 50 °C (no residual activity to interfere with subsequent PCR); compatible with one-tube carryover prevention workflow; derived from cold-adapted organism enabling low-temperature inactivation. |
| Purity | >= 95% by SDS-PAGE. Single predominant band. |
| Concentration | 1 U/uL standard. Higher concentrations available. |
| Activity / Unit Definition | 1 unit releases 1 pmol uracil from poly(dA)·poly([³H]-dU) substrate in 60 min at 37 °C. |
| Molecular Weight | ~25 kDa (monomer). |
| Source / Origin | Recombinant enzyme expressed in E. coli. Gene sequence derived from a psychrophilic marine bacterium, codon-optimized and engineered for recombinant production. |
| pH Range / Optimal pH | Optimal pH 7.5-8.5. Active range pH 6.5-9.5. |
| Shipping Conditions | Shipped with cold packs to maintain below 10 °C. |
| Expiration Date / Stability | 24 months at -20 °C. Avoid exposure to temperatures above 25 °C for extended periods as gradual inactivation may occur. |
| Regulatory / Compliance | ISO 9001 certified manufacturing. Suitable for IVD kit incorporation with carryover prevention feature. |
| Compatibility | Fully compatible with hot-start Taq and antibody-mediated DNA polymerases. Compatible with SYBR Green, TaqMan probe, and molecular beacon detection chemistries. dUTP/dNTP mix optimized for replacement of dTTP in PCR. |
| Recommended Buffer System | Compatible with standard Tris and HEPES PCR buffers. Optimal in pH 7.5-8.5 with 1-5 mM Mg²⁺. Not inhibited by common PCR buffer additives. |
| Application Notes / Precautions | For carryover prevention: use dUTP in place of dTTP in all PCR reactions. Add UDG directly to PCR master mix. Pre-incubation at 37 °C for 10 min digests carryover products. Caution: dUTP-containing PCR products may migrate differently on agarose gels. Thaw UDG on ice and return to -20 °C promptly after use. |
| Batch-to-Batch Consistency | Activity and heat-labile inactivation characteristics consistent across production lots. Carryover digestion efficiency >99.9% verified per batch. |
For research use only, not for clinical use.
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