Description
Exonuclease I (Exo I) is an exonuclease that hydrolyzes single-stranded DNA, one nucleotide at a time from the end, in the 3´→5´ direction. It releases 5´- mononucleotides one after another and leaves the terminal 5´-dinucleotide intact. This recombinant enzyme, does not cleave DNA strands without terminal 3´-hydroxyl groups, as these are blocked by phosphoryl or acetyl groups. It does not degrade double-stranded DNA. For activity, Exo I requires magnesium (optimal [Mg2+] is 10mM). Exo I is tolerant to a wide variety of buffer conditions (salt, pH, etc), and thus can be added directly to most molecular biology buffers containing magnesium (>1mM), including PCR reaction mixtures.
Features
Quantity and Specifications
Specific Activity: 20U/μl. Purified from a recombinant E.coli strain harbouring the sbcB gene. Product comprises of 20.000U of Exonuclease I in 1ml of 10mM Tris-HCl pH7,5 (25ºC), 100mM NaCl, 1mM DTT, 0.5mM EDTA and 50% glycerol.
Units
One Unit (1 U) is defined as the amount of enzyme required to catalyze the release of 10nmol of acid-soluble nucleotides from .17mg/ml ssDNA in 50μl 1x reaction buffer during incubation at 37ºC in 30 minutes.
Storage and Shelf Life
Exo I can be stored for up to 2 years at -20ºC.
Inhibition
Exonuclease I can tolerate a wide variety of buffers and only a few common molecular biology compounds are known to inhibit Exo I. PEG8000 at high concentrations (20% (w/v)) inhibits Exonuclease I due to molecular crowding.
Inactivation
Exonuclease I can be heat-inactivated by incubation at 80ºC for 1 minute or at 60ºC for 15 minutes.
Applications
Removal of primers from PCR reaction mixtures prior to DNA sequencing or DNA labelling (typically in a PCR Clean-up protocol in combination with the use of Shrimp Alkaline Phosphatase (SAP)). Removal of linear DNA molecules from plasmid preparations (in combination with the use of Lambda Exonuclease). Removal of ssDNA containing a 3´-hydroxyl group from heterogeneous mixtures of
nucleic acids. Assay for the presence of ssDNA regions.
