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 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.
Molecular Devices Corporation, Menlo Park, California 94025.
V T
VT
Kung
P R
PR
Panfili
E L
EL
Sheldon
R S
RS
King
P A
PA
Nagainis
B
B
Gomez
D A
DA
Ross
J
J
Briggs
R F
RF
Zuk
D000818:Animals; D002417:Cattle; D006224:Cricetinae; D003412:Cricetulus; D004277:DNA, Single-Stranded / Q000032:analysis*; D004268:DNA-Binding Proteins; D005374:Filtration; D006863:Hydrogen-Ion Concentration; D051379:Mice; D009691:Nucleic Acid Denaturation; D012680:Sensitivity and Specificity; D012825:Silicon; D014510:Urease
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D014510:Urease; D012825:Silicon