Exonuclease I
Useful in preparing PCR products for applications involving sequencing or labeling methods
Source:
E. coli strain containing an overproducing clone of E. coli Exonuclease I.
Description:
Exonuclease I hydrolyzes single-stranded DNA in the 3'→5' direction, releasing
5'-mononucleotides and leaving the terminal 5'-dinucleotide intact. Hydrolysis is processive and cannot proceed if the 3' terminus is phosphorylated. Exonuclease I can be used to measure the endonucleolytic cleavage of covalently closed circular single-stranded DNA reacted with an endonuclease of interest(1). In addition, DNA helicase activity can be measured utilizing Exonuclease I(2).
Exonuclease I is particularly useful in preparing the products of PCR for applications involving sequencing or labeling methods(3). Typically, the excess primers and any other extraneous single-stranded DNA present in PCR products will interfere with subsequent enzymatic reactions involving DNA synthesis. The hydrolytic properties of Exonuclease I degrade all single-stranded DNA present in the PCR mixture allowing the product to be used more efficiently in other applications. When combined with Shrimp Alkaline Phosphatase (PN 70092) for dNTP dephosphorylation, the use of alternative purification methods, such as columns, gels or magnetic separations, are completely eliminated.
For PCR clean up with Exonuclease I, see the USB ExoSAP-IT protocol. The purchase of ExoSAP-IT provides a license to the methods of PCR clean up using Exonuclease I and SAP.
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Properties:
Molecular Weight: 55 kDa
Heat Inactivation: 80° for 15 min.
Degrades to terminal dinucleotides.
Degrades glycosylated DNA.
Optimum Temperature: 37°C
Purity:
Greater than 95% pure as determined by SDS-PAGE. Tested for contaminating endonucleases, double-stranded exonucleases and ribonucleases.
Storage Buffer:
20mM Tris-HCI (pH 7.5), 5mM 2-mercaptoethanol, 0.5mM EDTA, 50% glycerol.
Assay Conditions:
The reaction mixture (100 μl) contains 67mM glycine buffer (pH 9.5), 10mM
2-mercaptoethanol, 6.7mM MgCl2, 0.5mM denatured DNA and enzyme. Incubation is at 37°C for 30 min.
Unit Definition:
One unit is the amount of enzyme which catalyzes the release of 10 nmol of acid-soluble nucleotide from denatured DNA in 30 min at 37°C under standard conditions.
Concentration:
Standard Conc.: 10 units/μl, PN 70073
High Conc.: 20 units/μl, PN 72073
Functional Assay: Treated PCR product with Exonuclease I to degrade unincorporated primers before performing sequencing reaction with Sequenase™ Version 2.0 DNA Polymerase Sequencing Kit (PN 70170).
References:
- GOLDMARK, P. J. AND LINN, S. (1972) J. Biol. Chem. 247, 1849-1860.
- ROSAMOND, J., TELANDER, K. M. AND LINN, S. (1979) J. Biol. Chem. 254, 8646-8652.
- WERLE, E., SCNEIDER C., RENNER, M., VÖLKER, M. AND FIEHN, W. (1994) Nucleic Acids Res. 22, 4354-4355.
- HANKE, M. AND WINK, M. (1994) BioTechniques 17, 858-860.
Applications:
- Elimination of residual single-stranded DNA containing a 3' terminus.
- Measuring endonucleolytic cleavage of covalently closed circular ssDNA.
- Measuring DNA helicase activity.
Storage:
Shipped on dry ice. Store at -20°C.