Picogram quantitation of total DNA using DNA-binding proteins in a silicon sensor-based system

We report a rapid and reproducible method to quantify total DNA at picogram levels. Two high-affinity DNA-binding proteins are used to construct a sandwich assay and a semiconductor sensor is used for quantitation. Single-stranded DNA-binding protein (SSB) from Escherichia coli is conjugated with a...

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Veröffentlicht in:Analytical biochemistry 1990-06, Vol.187 (2), p.220-227
Hauptverfasser: Kung, Viola T., Panfili, Peter R., Sheldon, Edward L., King, Robert S., Nagainis, Peter A., Gomez, Baltazar, Ross, Debra A., Briggs, Jonathan, Zuk, Robert F.
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Sprache:eng
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Zusammenfassung:We report a rapid and reproducible method to quantify total DNA at picogram levels. Two high-affinity DNA-binding proteins are used to construct a sandwich assay and a semiconductor sensor is used for quantitation. Single-stranded DNA-binding protein (SSB) from Escherichia coli is conjugated with a linker molecule, biotin, for specific capture of the DNA complex onto a membrane. Monoclonal anti-DNA antibody is conjugated with an enzyme, urease, for signal generation. To detect DNA, a sample is denatured to form single-stranded DNA and then incubated with a reagent containing both DNA-binding protein conjugates and streptavidin. After incubation of the reagent with the DNA sample for 1 h at 37°C to form a complex of streptavidin-biotin-SSB-DNA-anti-DNA-urease, the mixture is filtered through a biotin-coated nitrocellulose membrane which binds the streptavidin component of the complex. The unbound reagent is washed off the membrane, and then the captured DNA complex is detected with a ligh-addressable potentiometric sensor which measures the pH change catalyzed by the urease in the complex. This assay can detect 2 pg of DNA with a quantitation coefficient of variation of less than 10% in the range 10 to 200 pg.
ISSN:0003-2697
1096-0309
DOI:10.1016/0003-2697(90)90447-H