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Detection of total DNA with single-stranded DNA binding protein conjugates
E L Sheldon1, P A Nagainis, V T Kung
1Molecular Devices Corporation, Menlo Park, California 94025.
Biochemical and Biophysical Research Communications
|November 30, 1989
Summary
A new DNA detection method uses E. coli single-stranded DNA binding protein for rapid and sensitive total DNA measurement. This assay can detect DNA contamination in therapeutic proteins within 3 hours.
Area of Science:
- Biochemistry
- Molecular Biology
- Analytical Chemistry
Background:
- Accurate DNA quantification is crucial in molecular biology and biotechnology.
- Existing methods for DNA measurement may lack speed or sensitivity.
- Detecting DNA contamination in biopharmaceuticals is essential for safety and efficacy.
Purpose of the Study:
- To develop a rapid and sensitive assay for total DNA measurement.
- To utilize single-stranded DNA binding protein (SSB) enzyme conjugates for DNA detection.
- To apply this method for detecting DNA contamination in therapeutic proteins.
Main Methods:
- DNA denaturation via heat or alkali treatment.
- Immobilization of single-stranded DNA (ssDNA) onto nylon or nitrocellulose membranes.
- Incubation with E. coli SSB protein conjugated with horseradish peroxidase or urease.
- Colorimetric detection of bound enzyme-conjugate after washing.
Main Results:
- The assay demonstrates high sensitivity, detecting as little as 10 pg of DNA.
- The method provides rapid results, completing in under 3 hours.
- Successful application in detecting DNA contamination in therapeutic proteins.
Conclusions:
- A novel, rapid, and sensitive DNA measurement method has been established.
- The SSB enzyme conjugate assay is effective for total DNA quantification.
- This technique is valuable for quality control in biopharmaceutical production, ensuring the absence of DNA contaminants.