Endonuclease VIII (10000 U)

QIAGEN's recombinant Endonuclease VIII is an E. coli-derived enzyme that functions as both an N-glycosylase and an AP-lyase. It excises oxidative base lesions to generate an AP site, then cleaves the phosphodiester backbone, leaving terminal phosphates at both the 5ʹ and 3ʹ ends.

Damaged bases recognised include urea, 5,6-dihydroxythymine, thymine glycol, 5-hydroxy-5-methylhydanton, uracil glycol, 6-hydroxy-5,6-dihydrymine and methyltartronylurea. The enzyme is supplied in 10 mM Tris-HCl, 250 mM NaCl, 0.1 mM EDTA and 50% glycerol at pH 8.0 (25°C), and the 10x reaction buffer comprises 100 mM Tris-HCl, 750 mM NaCl and 10 mM EDTA at pH 8.0 and 25°C. One unit 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 specifications include a specific activity of 770,000 U/mg and physical purity of >99% as assessed by SDS-PAGE with silver-stain detection. Functional assays confirm <1% released from a single-stranded exonuclease test (10 U), <1% released from a double-stranded endonuclease test (100 U), and <10 copies of E. coli genomic DNA contamination (100 U). Activity is measured by two-fold serial dilution using a FAM-labeled 34-base duplex oligonucleotide containing a single uracil, pre-treated with UDG to create an abasic site.