Uracil Cleavage System (750 rxn)

For creating an abasic site with an intact phosphodiester backbone and liberating the deoxyribose sugar, the Uracil Cleavage System from QIAGEN provides two enzymes, Uracil DNA Glycosylase (UDG) and Endonuclease VIII, supplied at a 10x concentration. When added sequentially to a reaction containing a synthetic DNA fragment containing a deoxy-uracil, these enzymes generate a single nucleotide gap at the location of the uracil residue. UDG catalyzes the excision of the uracil base, creating an abasic site with an intact phosphodiester backbone. The lyase activity of Endonuclease VIII then breaks the phosphodiester backbone, both 3' and 5' to the abasic site, liberating the deoxyribose sugar.

UDG is supplied in 10 mM Tris-HCl, 50 mM NaCl, 1 mM DTT, 0.1 mM EDTA and 50% glycerol (pH 7.5 at 25°C). Endonuclease VIII is supplied in 10 mM Tris-HCl, 250 mM NaCl, 0.1 mM EDTA and 50% glycerol (pH 8.0 at 25°C). The proteins are produced and purified separately from E. coli strains containing recombinant Endonuclease VIII and Uracil-DNA Glycosylase genes. The enzymes are active in most molecular biology reaction buffers, so there is no need to exchange buffers before assembling the cleavage reaction. For a 10 µL reaction, add 0.5 µL UDG and 0.5 µL Endonuclease VIII to DNA substrate (final concentration up to 300 nM) in 1x reaction buffer, then incubate at 37°C for 30 minutes. Following incubation, the reaction can be stopped by adding a gel-loading stop solution in preparation for electrophoretic analysis or prepared for transformation.

One unit of UDG is defined as the amount of enzyme that catalyzes the release of 1.8 nmol of uracil in 30 minutes from double-stranded, tritiated, uracil-containing DNA at 37°C in 1x UDG Reaction Buffer. One unit of Endonuclease VIII is defined as the amount of enzyme required to cleave 1 pmol of an oligonucleotide duplex containing a single AP site in 1 hour at 37°C. Quality control specifications include: Purity >99%, single-stranded exonuclease <5% released, double-stranded exonuclease <1% released, double-stranded endonuclease activity showing no conversion, and E. coli DNA contamination <10 copies.