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| Product Name | High-Fidelity DNA Polymerase (Proofreading) |
| Catalog No. | IEC-HMM-0049 |
| Description | A recombinant DNA polymerase engineered for high-fidelity PCR amplification, incorporating 3'-5' exonuclease proofreading activity that corrects misincorporated nucleotides during synthesis. This enzyme exhibits fidelity approximately 50-fold higher than Taq DNA polymerase, making it suitable for applications where sequence accuracy is paramount. |
| Intended Use | Intended for high-fidelity PCR amplification of templates up to 15 kb for downstream cloning, mutagenesis, next-generation sequencing library preparation, and applications requiring extremely low error rates. |
| Principle / Technology | Combines a thermostable DNA polymerase domain with an associated 3'-5' exonuclease proofreading domain. During polymerization, mispaired nucleotides are recognized and excised by the proofreading domain before chain extension continues, dramatically reducing the overall error frequency. |
| Detection Method | Polymerase activity measured by dNTP incorporation assay. Fidelity assessed by lacI forward mutation assay or rpsL-based reversion assay to determine error frequency per base per duplication. |
| Sample Type | Genomic DNA (high molecular weight), plasmid and cosmid DNA, cDNA, and long-range PCR templates. Not recommended for colony PCR or templates containing PCR inhibitors. |
| Performance Range / Specifications | Fidelity: ~50x higher than Taq (~4.4 x 10⁻⁷ errors/base/duplication). Extension rate: ~15-30 s/kb. Amplicon range: 200 bp to 15 kb. Generates blunt-ended products (no 3'-A overhang). |
| Sensitivity / LOD | Reliably amplifies single-copy targets from 1-10 ng human genomic DNA after 30 cycles. |
| Specificity | High specificity due to proofreading activity rejecting mismatched primer-template duplexes. Primer design should include GC clamps at the 3' end for optimal discrimination. |
| Reaction Conditions / Protocol | Initial denaturation: 98 °C, 30 s. Cycling (25-35x): 98 °C 10 s, 55-72 °C 15 s, 72 °C 15-30 s/kb. Final extension: 72 °C 5 min. Annealing temperature typically 3-5 °C above standard Taq protocols. |
| Components / Formulation | High-fidelity DNA Polymerase (2 U/uL) in 20 mM Tris-HCl (pH 7.5), 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 50% glycerol, 0.1% Triton X-100, 0.1% Tween-20. Supplied with 5x HF Buffer containing optimized Mg²⁺. |
| Storage Conditions | Store at -20 °C to -30 °C. Minimize freeze-thaw cycles. |
| Shelf Life | 24 months at -20 °C. |
| Package Specifications | 100 U (50 uL), 500 U (250 uL), 2,500 U (1.25 mL). Includes 5x HF Buffer and optional DMSO (for GC-rich templates). |
| Product Form | Clear, colorless liquid in glycerol-based storage buffer. |
| Quality Control | Each lot tested for: specific activity; proofreading fidelity; absence of contaminating DNase, RNase, and endonuclease; functional amplification of templates 0.5-15 kb; Sanger sequencing verification of cloned products. |
| Key Features | ~50x fidelity improvement over Taq; generates blunt ends for direct ligation cloning; amplifies up to 15 kb; suitable for NGS library amplification with minimal sequence bias. |
| Purity | >= 95% homogeneity by SDS-PAGE with Coomassie staining. |
| Concentration | 2 U/uL standard. Also available at 1 U/uL and 5 U/uL concentrations for specialized applications. |
| Activity / Unit Definition | 1 unit incorporates 10 nmol dNTP in 30 min at 74 °C using activated calf thymus DNA. Proofreading activity verified by mutation frequency assay. |
| Molecular Weight | ~90 kDa (polymerase subunit). Full complex molecular weight depends on the specific enzyme architecture (single-chain or fusion protein). |
| Source / Origin | Recombinant enzyme produced in E. coli. Polymerase gene sequence derived from hyperthermophilic archaeal species and optimized through protein engineering for enhanced processivity and fidelity. |
| pH Range / Optimal pH | Optimal pH 7.5-8.5. Activity declines rapidly below pH 7.0 or above pH 9.0. |
| Shipping Conditions | Shipped with cold packs to maintain below 10 °C. Once received, transfer immediately to -20 °C storage. |
| Expiration Date / Stability | 24 months at -20 °C. Avoid repeated freeze-thaw cycles; prepare working aliquots on receipt. |
| Regulatory / Compliance | ISO 9001 certified production. For research use. IVD raw material supply with regulatory documentation available. |
| Compatibility | Compatible with standard thermal cyclers. Blunt-end products require A-tailing or blunt-end cloning vectors. Not recommended when 3'-A overhangs are required for TA cloning. |
| Recommended Buffer System | 5x HF Buffer: proprietary formulation with optimized Mg²⁺ concentration and enhancers. GC-rich buffer variant available for templates >65% GC content. |
| Application Notes / Precautions | Use 3-5 °C higher annealing temperature than with Taq. For long amplicons (>5 kb), increase extension time to 30-60 s/kb. Add DMSO (3-5%) for templates exceeding 65% GC content. Do not use for TA cloning unless an A-tailing step is performed. |
| Batch-to-Batch Consistency | Fidelity verified per production lot by mutation frequency assay. Lot-to-lot activity variation <= +/- 10%. |
For research use only, not for clinical use.
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