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PMID: 3748805 Published · ppublish English Journal Article

Preparation of oligodeoxynucleotide-alkaline phosphatase conjugates and their use as hybridization probes.

Nucleic acids research ·Vol. 14 ·No. 15 ·1986-08-11 ·Pages 6115-28

Jablonski E, Moomaw EW, Tullis RH, Ruth JL

Abstract

Short synthetic oligonucleotides have been covalently cross-linked to alkaline phosphatase using the homobifunctional reagent disuccinimidyl suberate. The oligomers, twenty-one to twenty-six bases in length, are complementary to unique sequences found in herpes simplex virus, hepatitis B virus, Campylobacter jejuni and enterotoxigenic Escherichia coli. Each oligomer contains a single modified base with a 12-atom "linker arm" terminating in a reactive primary amine. Cross-linking through this amine results in oligomer-enzyme conjugates composed of one oligomer per enzyme molecule that have full alkaline phosphatase activity and can hybridize to target DNA fixed to nitrocellulose within 15 minutes. The hybrids are detected directly with a dye precipitation assay at a sensitivity of 10(6) molecules (2 X 10(-18) mol) of target DNA in 4 hours development time. The enzyme has no apparent effect on selectivity or kinetics of oligonucleotide hybridization and the conjugates can be hybridized and melted off in a conventional manner.

MeSH Terms
Alkaline Phosphatase/metabolism Base Sequence Cross-Linking Reagents Methods Nucleic Acid Hybridization Oligodeoxyribonucleotides/chemical synthesis,metabolism Protein Binding Succinimides
Chemicals
Cross-Linking Reagents Oligodeoxyribonucleotides Succinimides Alkaline Phosphatase disuccinimidyl suberate
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Jablonski E
Moomaw E W
Tullis R H
Ruth J L
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33 references, click to expand
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Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
0305-1048
DOI
10.1093/nar/14.15.6115
Published
1986-08-11
Pages
6115-28
Language
English
Region
England
NLM ID
0411011
PMCID
PMC311625
Subset
IM
External Links
PubMed source DOI: 10.1093/nar/14.15.6115 PMC Full Text View journal details
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