Synthesis of 5′-oligonucleotide hydrazide derivatives and their use in preparation of enzyme-nucleic acid hybridization probes
| Autor: | Deborah Y. Kwoh, Philip M. Kao, Soumitra S. Ghosh |
|---|---|
| Rok vydání: | 1989 |
| Předmět: |
Male
Ion chromatography Biophysics Hydrazone Hydrazide Biochemistry High-performance liquid chromatography chemistry.chemical_compound Nucleic acid thermodynamics Animals Organic chemistry Molecular Biology Chromatography High Pressure Liquid chemistry.chemical_classification Chemistry Oligonucleotide Cell Biology Alkaline Phosphatase Enzymes Immobilized Combinatorial chemistry Rats Hydrazines Nucleic acid Spectrophotometry Ultraviolet Oligonucleotide Probes Molecular probe |
| Zdroj: | Analytical Biochemistry. 178:43-51 |
| ISSN: | 0003-2697 |
| DOI: | 10.1016/0003-2697(89)90354-0 |
| Popis: | A hydrazone-based method for conjugating synthetic nucleic acids and reporter molecules for use as nonradioactive hybridization probes is presented. Oligonucleotides complementary to the hepatitis B virus were derivatized at their 5′ ends with hydrazine or homobi-functional acyl hydrazides. These derivatives reacted facilely with aldehydes to give hydrazones, which were characterized by uv spectroscopy and HPLC. Coupling of aldehyde-modified alkaline phosphatase with carbohydrazide-oligonucleotide derivatives provided a mixture of two enzyme-nucleic acid conjugates in 80–85% yield. The conjugates had a 1:1 and a 2:1 oligonucleotide/enzyme ratio, respectively, and were separated by ion-exchange chromatography. Both conjugates were able to detect 7 amol of target DNA in 1 h, using a colorimetric assay. In contrast, oligonucleotide-horseradish peroxidase conjugates were 40-fold lower in sensitivity of detection. |
| Databáze: | OpenAIRE |
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