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| Product Name | Exonuclease III (100 U/μL) |
| Catalog No. | IVDRM-0236 |
| Description | Exonuclease III is an exonuclease that acts on double-stranded DNA, catalyzing the stepwise removal of mononucleotides from 3'-OH termini. The preferred substrates are blunt-ended or 5'-overhang DNA, although the enzyme can also act at nick sites in duplex DNA to generate single-stranded gaps. Because it is inactive on single-stranded DNA, it is essentially unable to cleave 3'-overhang termini; the extent of digestion by the 3'→5' exonuclease activity decreases with the length of the 3' overhang, and overhangs of four bases or longer are resistant to cleavage. This property can be exploited to generate single-stranded DNA in a defined direction: by designing linearized DNA with a cleavage-resistant terminus (3' overhang) at one end and a susceptible terminus (blunt or 5' overhang) at the other, Exonuclease III will digest only one strand. Exonuclease III activity partially depends on the DNA duplex structure and varies with sequence (C > A = T > G). The enzyme also exhibits RNase H, 3'-phosphatase, and apurinic/apyrimidinic (AP) site-specific endonuclease activities. |
| Intended Use | Raw material enzyme for molecular biology and diagnostic applications requiring controlled 3'→5' digestion of double-stranded DNA, including directional single-stranded DNA generation, strand-specific DNA modification, and use as a component of RPA (recombinase polymerase amplification) workflows. |
| Principle / Technology | Exonuclease III catalyzes the stepwise hydrolysis of mononucleotides from 3'-OH termini of double-stranded DNA. Its strong preference for duplex substrates (blunt ends, 5' overhangs, or internal nicks) and inactivity toward single-stranded DNA and 3' overhangs of four bases or more allow selective, directional digestion of one DNA strand. |
| Performance Range / Specifications | - Preferred substrates: blunt-ended or 5'-overhang double-stranded DNA; also acts at nick sites to generate single-stranded gaps. - No activity on single-stranded DNA; 3'-overhang termini are resistant to cleavage. - Digestion decreases with 3'-overhang length; overhangs of four bases or longer are essentially not cleaved. - Additional activities: RNase H, 3'-phosphatase, and AP site-specific endonuclease. |
| Reaction Conditions / Protocol | Typical digestion: incubate double-stranded DNA substrate with Exonuclease III at 37 °C for 30 min. The reaction can be stopped by adding EDTA (e.g., 5.5 mM final concentration) to inactivate the enzyme. In performance testing, 1 μg of λDNA in a 50 uL reaction was completely degraded at low enzyme input (0.06–20 U range tested) following 30 min incubation at 37 °C. |
| Storage Conditions | Store at -25 °C to -15 °C. |
| Shelf Life | 2 years |
| Package Specifications | 5,000 U 25,000 U |
| Concentration | 100 U/uL |
| Expiration Date / Stability | Stable for 2 years when stored at -25 °C to -15 °C. |
| Application Notes / Precautions | - To generate single-stranded DNA in a defined orientation, design the linearized substrate with a cleavage-resistant 3'-overhang terminus at one end and a susceptible (blunt or 5'-overhang) terminus at the other; only one strand will be digested. - Avoid substrates with 3' overhangs of four bases or longer if complete digestion is required, as these termini are resistant to the enzyme. - Enzyme activity is influenced by DNA sequence context (C > A = T > G); optimize enzyme input empirically for each substrate. - The enzyme can be inactivated by chelating divalent cations with EDTA. |
| Recognition Site | Acts at 3'-OH termini of double-stranded DNA (blunt ends, 5' overhangs, or nick sites); no defined sequence recognition site, though activity varies with sequence context (C > A = T > G). |
For research use only, not for clinical use.
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