Patent ID: 7172861
Filing Date: 2007-02-06
Classification: C12Q,G01N

Abstract:
1. A method for detecting N-glycosylase activity comprising: (a) providing an oligodeoxyribonucleotide substrate comprising (1) a 5′ segment, (2) a deoxyadenosine residue comprising an adenine residue covalently bonded to a 2-deoxyribose residue through a β-linkage, or a deoxyuridine residue comprising a uracil residue covalently bonded to a 2-deoxyribose residue through a β-linkage, and (3) a 3′ segment, which lies 3′ to the deoxyadenosine or deoxyuridine residue, (b) inactivating the N-glycosylase activity in the mixture to result in an N-glycosylase-inactivated mixture; (c) treating the N-glycosylase product and any substrate that may be present in the N-glycosylase-inactivated mixture with an oligodeoxyribonucleotide primer under conditions such that a limited extension product of the primer is synthesized and, if the substrate is present in the mixture, a longer extension product of the primer is synthesized, (d) separating the limited and longer extension products from the N-glycosylase product and the substrate, respectively, on which they were synthesized to produce corresponding single-stranded molecules; (e) treating the single-stranded molecules generated from step (d) under conditions such that additional limited and longer primer extension products are synthesized using the N-glycosylase product and any substrate that may be present as templates, and the limited primer extension products form hairpin structures and are further extended, using the 5′ portion as template, such that a label is incorporated to result in a labeled product, but the longer primer extension products do not substantially incorporate the label; and (f) separating and detecting the labeled product, thereby detecting N-glycosylase activity.