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A highly sensitive and fast nonradioactive method for detection of polymerase chain reaction products
K Holmstrøm1, L Rossen, O F Rasmussen
1Department of Molecular Food Technology, Biotechnological Institute, Lyngby, Denmark.
Abstract:
A novel system for the detection of polymerase chain reaction (PCR) products has been developed. The system is based on a PCR in which one of the primers is biotinylated and digoxigenin-11-dUTP is incorporated during elongation. Biotinylated PCR products are captured on streptavidin-coated solid supports, and alkaline phosphatase conjugated to anti-digoxigenin antibody is subsequently bound to the incorporated digoxigenin. The detection may be obtained with colorimetric, fluorescent, or luminescent substrates for alkaline phosphatase. The detection system can be performed in microtiter plates allowing easy handling of multiple samples. The total assay time following the PCR is between 1 and 2 h dependent on the type of substrate and the type of solid support applied in the system. Within this period of time the system is capable of detecting 1 template in 29 cycles of PCR.