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Title: Activity-based assay for ricin-like toxins

Abstract

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.

Inventors:
;
Issue Date:
Research Org.:
Idaho National Lab. (INL), Idaho Falls, ID (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1176096
Patent Number(s):
7172861
Application Number:
10/944,259
Assignee:
Battelle Energy Alliance, LLC (Idaho Falls, ID)
Patent Classifications (CPCs):
G - PHYSICS G01 - MEASURING G01N - INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
C - CHEMISTRY C12 - BIOCHEMISTRY C12Q - MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS
DOE Contract Number:  
AC07-99ID13727
Resource Type:
Patent
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES

Citation Formats

Keener, William K., and Ward, Thomas E.. Activity-based assay for ricin-like toxins. United States: N. p., 2007. Web.
Keener, William K., & Ward, Thomas E.. Activity-based assay for ricin-like toxins. United States.
Keener, William K., and Ward, Thomas E.. Tue . "Activity-based assay for ricin-like toxins". United States. https://www.osti.gov/servlets/purl/1176096.
@article{osti_1176096,
title = {Activity-based assay for ricin-like toxins},
author = {Keener, William K. and Ward, Thomas E.},
abstractNote = {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.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = {2007},
month = {2}
}

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Works referenced in this record:

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Gelonin Is an Unusual DNA Glycosylase That Removes Adenine from Single-stranded DNA, Normal Base Pairs and Mismatches
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