QUANTIFICATION OF POLYMERASE CHAIN-REACTION PRODUCTS - ENZYME-IMMUNOASSAY BASED SYSTEMS FOR DIGOXIGENIN-LABELED AND BIOTIN-LABELED PRODUCTSTHAT QUANTIFY EITHER TOTAL OR SPECIFIC AMPLICONS

Enzyme immunoassays were developed for quantification of polymerase ch ain reaction (PCR) products, referred to as amplicons. Amplicons were dual labelled simultaneously by enzymatic incorporation of digoxigenin and biotin during PCR. For total amplicon quantification, Microfluor B polystyrene wells, compatible with chemiluminescent detection, were coated with streptavidin. Dual labelled amplicons were bound, treated with anti-digoxigenin antibody conjugated to alkaline phosphatase to c omplete the two-site sandwich immunoassay configuration, and detected by the chemiluminescence generated upon hydrolysis of a phosphate subs tituted dioxetane substrate, AMPPD. For specific amplicon quantificati on, the Microfluor B wells were coated with an unlabelled DNA probe co mplementary to the labelled amplicon target. Subsequent steps were per formed as described above. This assay detects 2 pg of specifically amp lified DNA. Chemiluminescent detection provides a linear range of four orders of magnitude for amplicon quantification. The non-radioactive labels are safe and stable. PCR as described here obviates the need fo r labelled primers and constitutes the initial report of concurrent du al non-radioactive labelling of DNA by a DNA polymerase. (C) 1996 Acad emic Press Limited