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Activity-based assay for ricin-like toxins

United States Patent

February 6, 2007
View the Complete Patent at the US Patent & Trademark Office
Idaho National Laboratory - Visit the Technology Transfer and Commercialization Office Website
A method of detecting N-glycosylase activity in a sample involves incubating an oligodeoxyribonucleotide substrate containing a deoxyadenosine or deoxyuridine residue with the sample to be tested such that the N-glycosylase, if present, hydrolyzes the deoxyadenosine or deoxyuridine residue to result in an N-glycosylase product having an abasic site. A primer is annealed to the N-glycosylase product, and the primer is extended with a DNA polymerase, such as Taq DNA polymerase, that pauses at abasic sites. The resulting extension products are melted from the N-glycosylase product, allowed to form hairpins due to self-complementarity, and further extended in the presence of labeled precursors to result in labeled products. Extension products synthesized from undigested substrate as template do not result in labeled products. Thus, detection of labeled products results in detection of N-glycosylase activity. Oligodeoxyribonucleotide substrates, primer, and positive controls and a kit for N-glycosylase assay are also disclosed.
Keener; William K. (Falling Waters, WV), Ward; Thomas E. (Pennsylvania Furnace, PA)
Battelle Energy Alliance, LLC (Idaho Falls, ID)
10/ 944,259
September 16, 2004
GOVERNMENT RIGHTS This invention was made with government support under Contract No. DE-AC07-99ID13727 with the Department of Energy. The government has certain rights in the invention.