HOMOGENEOUS DETECTION OF NUCLEIC-ACIDS BY TRANSIENT-STATE POLARIZED FLUORESCENCE

We describe a transient-state polarized fluorescence-based method for detecting nucleic acids. An active ester of the phthalocyanine dye La Jolla Blue(TM) was coupled to an oligonucleotide containing an amino g roup at its 5' end, and the conjugate was purified by HPLC chromatogra phy. We monitored the hybridization characteristics of the conjugate w ith complementary oligonucleotides and RNA as targets by transient-sta te polarized fluorescence measurements. The method was comparable in s ensitivity to isotopic and nonisotopic heterogeneous detection systems and was capable of detecting 1 fmol of a 382-base-long RNA transcript from human immunodeficiency virus type 1 (HIV-1) generated in a self- sustained sequence replication (3SR) reaction.