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:

Keener, William K.; Ward, Thomas E.

Issue Date:

2007-02-06

Research Org.:

Idaho National Laboratory (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):

C - CHEMISTRY C12 - BIOCHEMISTRY C12Q - MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS

G - PHYSICS G01 - MEASURING G01N - INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES

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C - CHEMISTRY C12 - BIOCHEMISTRY C12Q - MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS

C12Q1/34 - involving hydrolase

G - PHYSICS G01 - MEASURING G01N - INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES

G01N2333/924 - acting on glycosyl compounds

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DOE Contract Number:  

AC07-99ID13727

Resource Type:

Patent

Country of Publication:

United States

Language:

English

Subject:

60 APPLIED LIFE SCIENCES