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 singlestranded 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 20×106 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 1·2×106 and 4·0×102 when the added quantities were 3×106 and 3×103, as determined by serial dilution. © 1991 Academic Press Ltd.