A System for the Quantitation of DNA Using a Microtiter Plate-Based Hybridization and Enzyme Amplification Technology
| Autor: | S. Harbron, Mark Fisher, Christopher John Taylorson |
|---|---|
| Rok vydání: | 1997 |
| Předmět: |
Biophysics
Biochemistry Substrate Specificity law.invention Glucose Oxidase Microtiter plate chemistry.chemical_compound Plasmid law Humans Molecular Biology Polymerase chain reaction chemistry.chemical_classification Nuclease Oxidase test biology Microchemistry Single-Strand Specific DNA and RNA Endonucleases Nucleic Acid Hybridization DNA Ribonuclease Pancreatic Cell Biology Hydrogen-Ion Concentration Molecular biology Enzyme chemistry biology.protein Colorimetry Pancreatic ribonuclease Plasmids |
| Zdroj: | Analytical Biochemistry. 251:280-287 |
| ISSN: | 0003-2697 |
| DOI: | 10.1006/abio.1997.2270 |
| Popis: | A quantitative hybridization technique for the detection of plasmid DNA by the action of a nuclease enzyme is described. The process utilizes the specific capture and detection of a sandwich hybridization, in a microtiter plate, that occurs in a single step. The detector probe is labeled with nuclease P 1 . The pH-dependent specificity of this enzyme for 3′-dinucleotides is used to generate a measurable signal by activating apo-glucose oxidase, which triggers an enzyme amplification cascade in the same microtiter plate. The sensitivity of the assay system is demonstrated in an assay of a mutated form of the human pancreatic ribonuclease gene inserted into the plasmid pUC 18. The system was able to detect 35 amol of target DNA in an assay composed of a 60-min hybridization and 20 min of signal generation. This use of nuclease P 1 as the enzyme label and apo-glucose oxidase as the trigger for the amplification cascade results in an assay that is more sensitive than previously described enzyme amplification systems using colorimetric detection. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
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