Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:6.3.4.6 (urease)
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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 degrees 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 light-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.
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PMID:Picogram quantitation of total DNA using DNA-binding proteins in a silicon sensor-based system. 220 Mar 3