Quantitation of DNA hybridization in a silicon sensor-based system: application to PCR.
Rapid, quantitative hybridization assays with good sensitivity are needed in many applications, for example, determining the amount of specific product from PCR. We have developed an assay which relies on the hybridization of a biotinylated oligomer and a fluoresceinated oligomer to a single-stranded target in solution. The hybridized complex is captured by streptavidin to a biotinylated membrane. After capture, the hybridization complex is detected by an antifluorescein-urease conjugate which binds to the fluoresceinated probe. The membrane-bound urease conjugate is exposed to urea and assayed with a pH-sensitive silicon sensor. The total assay time is less than 2 h and the sensitivity limit is 20 x 10(6) molecules with a coefficient of variation, CV, of less than 10%. The assay was applied to the analysis of a model target using PCR. We were able to measure the amount of specific product and the amplification factor during the exponential phase of PCR. Using extrapolation from the measured amounts of amplified product, the initial amounts of target molecules were calculated to be 1.2 x 10(6) and 4.0 x 10(2) when the added quantities were 3 x 10(6) and 3 x 10(3), as determined by serial dilution.
Olson
J D
JD
Molecular Devices Corporation, Menlo Park, CA 94025.
Panfili
P R
PR
Zuk
R F
RF
Sheldon
E L
EL
D001710:Biotin / Q000737:chemistry; D003101:Collodion; D015342:DNA Probes / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000737:chemistry; D019793:Fluorescein; D005452:Fluoresceins / Q000737:chemistry; D005455:Fluorescent Antibody Technique; D006863:Hydrogen-Ion Concentration; D008567:Membranes, Artificial; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D012825:Silicon; D014159:Transducers; D014510:Urease / Q000378:metabolism
D015342:DNA Probes; D004277:DNA, Single-Stranded; D005452:Fluoresceins; D008567:Membranes, Artificial; D009838:Oligodeoxyribonucleotides; D001710:Biotin; D003101:Collodion; D014510:Urease; D019793:Fluorescein; D012825:Silicon