| abstract |
Combinations of polymerization, non-competitive hybridization and assay techniques is disclosed. In one aspect of the method, one member of a regular or anchored primer pair is modified to include a coupling agent capable of forming a tight bond (resistant to uncoupling in an alkaline denaturing environment) with a reactant. Competitive PCR is performed and the PCR products are coupled via the coupling agent to a reactant on the surface of a solid phase support. The bond between the reactant and the solid phase support in this and all embodiments is also resistant to uncoupling in an alkaline denaturing environment. In another aspect of the method, a primer is tightly coupled to the bound reactant and a polymerization of the competitor and target nucleic acids is performed on the solid phase. A third embodiment uses at least three primers, one of which is internal to the PCR templates and is bound to the solid phase support on which the entire PCR takes place. In the first two embodiments, sense strands are completely removed from solution with the bound antisense strands of the PCR products. Hybridization with sequence-specific probes is then performed in the absence of competition for binding by the sense strands. Sense strand removal and hybridization is optional in the third embodiment, where the bound three-primer PCR products can be detected by a detectible signal. Quantification of the target template may preferably be achieved in all embodiments by an enzyme-linked immunosorbent assay (ELISA), using ELISA data analysis software and standard curves. |