QUANTITATIVE POLYMERASE CHAIN-REACTION WITH ENZYME-LINKED-IMMUNOSORBENT-ASSAY DETECTION OF SELECTIVELY DIGESTED AMPLIFIED SAMPLE AND CONTROL DNA

A quantitative polymerase chain reaction (PCR) method for the exact qu antitation of DNA is described that does not require radioactive label ing or electrophoretic separation of product species, thereby avoiding hazardous and time-consuming procedures which have so far impeded rou tine 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 pair s which change a single restriction site for a new one. Thereby, it is possible to discriminate the two PCR product species by selective res triction enzyme digestion (RED). Because inner PCR primers were labele d with biotin and digoxigenin, respectively, nested PCR products can b e immobilized on avidin-coated microtiter plates and quantitated separ ately by enzyme-linked immunosorbent assay (ELISA) techniques. We demo nstrate 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 proced ure can be easily adapted for quantitation of other PCR products. It i s suitable for rapid and automatic screening of many samples in parall el, e.g., for detection and quantitation of pathogens, for quantitatio n of gene copy numbers or for gene expression after reverse transcript ion. (C) 1995 Academic Press, Inc.