QUANTITATIVE POLYMERASE CHAIN-REACTION USING A RECOMBINANT-DNA INTERNAL STANDARD AND TIME-RESOLVED FLUOROMETRY

Using a 308 bp DNA fragment (target DNA) as a template, we have synthe sized an internal standard (IS) that is of the same size and uses the same primers as the target but differs by a 26 bp centrally located se quence. We then designed quantitative polymerase chain reaction (PCR) assays in which the target DNA is coamplified with a constant amount o f IS (20 000 molecules). The presence of IS compensates for the reacti on-to-reaction variability of the amplification efficiency. The PCR pr oducts are assayed by two distinct hybridization protocols. The first approach (QPCR-1) requires that specific probes be immobilized onto mi crotiter wells, followed by hybridization with digoxigenin-labeled PCR product. In the second protocol (QPCR-2), PCR product is captured ont o the wells and hybridized with digoxigenin-tailed specific probes, In both assays, the hybrids are detected using an anti-digoxigenin-alkal ine phosphatase conjugate and 5'-fluorosalicylphosphate as substrate, The hydrolysis product forms a highly fluorescent complex with Tb3+-ED TA, as measured by time-resolved fluorometry. The ratio of the fluores cence values obtained for the amplified target DNA and IS is linearly related to the number of target DNA molecules present in the sample pr ior to amplification, The linear ranges are 1000-200 000 molecules for QPCR-1 and 2000-200 000 molecules for QPCR-2, The CVs ranged from 3.4 to 9.7%.