Quantitative Polymerase Chain Reaction with Enzyme-Linked Immunosorbent Assay Detection of Selectively Digested Amplified Sample and Control DNA

Abstract

A quantitative polymerase chain reaction (PCR) method for the exact quantitation of DNA is described that does not require radioactive labeling or electrophoretic separation of product species, thereby avoiding hazardous and time-consuming procedures which have so far impeded routine use of PCR, in particular in the clinical laboratory. Sample and internal control DNA are competitively amplified in a one-tube nested PCR. The control DNA differs from the sample DNA by only two base pairs which change a single restriction site for a new one. Thereby, it is possible to discriminate the two PCR product species by selective restriction enzyme digestion (RED). Because inner PCR primers were labeled with biotin and digoxigenin, respectively, nested PCR products can be immobilized on avidin-coated microtiter plates and quantitated separately by enzyme-linked immunosorbent assay (ELISA) techniques. We demonstrate here that with the combination of selective restriction enzyme digestion and ELISA (RED-ELISA) sample DNA ranging from nanomolar to attomolar concentrations can be quantitated within ±10%. This procedure can be easily adapted for quantitation of other PCR products. It is suitable for rapid and automatic screening of many samples in parallel, e.g., for detection and quantitation of pathogens, for quantitation of gene copy numbers or for gene expression after reverse transcription.