Fluorescence detection assay for homogeneous pcr hybridization systems

Abstract

An improved assay system for homogeneous assay detection and/or measuring target nucleic acid sequence replicated in a PCR amplification procedure is provided by employing a nucleic acid polymerase having 5' to 3' exonuclease activity and devoid of 3' to 5' exonuclease activity, oligonucleotide analytical probe blocked against chain extension at its 3' terminus and labeled at its 5' terminus with an energy transfer donor fluorophore, and oligonucleotide detection probe labeled at its 3' terminus with an energy transfer acceptor fluorophore, said probes being complements of each other but differing in nucleotide length so that their Tm's are at least 10.degree. C. apart, the Tm of the oligonucleotide analytical probe is equal to or greater than the Tm's of the oligonucleotide primers used in the PCR amplification procedure and the Tm of the oligonucleotide detection probe is lower than the temperature required to conduct the PCR thermal cycling steps of the PCR amplification procedure so that during the PCR thermal cyclic steps 5' fluorophore labeled nucleotide fragments are produced during the PCR extension phase by the 5' to 3' exonuclease activity of the nucleic acid polymerase on oligonucleotide analytical probe annealed to denatured strands of the target nucleic acid sequence. Detection or measurement of either (a) the 5' fluorophore labeled nucleotide fragments produced during the thermal cyclic steps and measured by fluorescence polarization or (b) oligonucleotide analytical probe hybridized to oligonucleotide detection probe measured spectrophotometrically by energy transfer measurement, provide a measure of the amount of oligonucleotide analytical probe used up in the amplification of the target nucleic acid sequence and thus provide a measure of amount of target nucleic acid sequence amplified in the PCR replication procedure.