Sensitive fluorescence-based thermodynamic and kinetic measurements of DNA hybridization in solution.

Kinetic and thermodynamic constants associated with DNA hybridization were determined in solution using fluorescence measurements and complementary fluorophore-labeled oligomers. One oligomer was labeled with a 5'-terminal fluorescein, and the other was labeled with a 3'-terminal rhodamine. The juxtaposition of the two labels in double-stranded complexes results in a strong quenching of the fluorescein emission, thereby providing the means for distinguishing single-stranded DNA from double-stranded DNA. Since measurements were based on fluorescence, DNA denaturation and association could be monitored routinely at strand concentrations 100-1000-fold lower than permitted by absorbance hypochromicity measurements. To determine if fluorescence quenching mirrored base pair formation, temperature profiles of DNA association and dissociation were constructed from both absorbance hypochromicity and fluorescence quenching measurements at a number of different DNA concentrations. Analyses of these profiles using the "all-or-none" model of hybridization provided thermodynamic data which were statistically indistinguishable between the two measurement methods, thus validating the use of fluorescence quenching in thermodynamic studies of oligomers. The effects of fluorophore attachment on the thermodynamic properties of the DNA strands were investigated by analyzing the melting curves of different combinations of unlabeled and labeled complementary oligomers. The presence of both labels was found to stabilize the double-stranded DNA by about -1.5 kcal in delta G degrees 298, primarily due to the fluorescein label. Association and dissociation rate constants were determined by fluorescence measurements at different temperatures, and linear Arrhenius plots were obtained. The fluorescence measurements provided a unique "label dilution" method for measuring dissociation rate constants of oligomers based upon the dynamic association and dissociation of complementary DNA strands at constant temperature. Association rate measurements were simplified since relatively low concentrations of complementary oligomers could be mixed, thereby reducing hybridization rates and eliminating the need for rapid mixing and measurement techniques.

Solution-phase detection of polynucleotides using interacting fluorescent labels and competitive hybridization.

DNA was assayed in a homogeneous format using DNA probes containing hybridization-sensitive labels. The DNA probes were prepared from complementary DNA strands in which one strand was covalently labeled on the 5'-terminus with fluorescein and the complementary strand was covalently labeled on the 3'-terminus with a quencher of fluorescein emission, either pyrenebutyrate or sulforhodamine 101. Probes prepared in this manner were able to detect unlabeled target DNA by competitive hybridization producing fluorescence signals which increased with increasing target DNA concentration. A single pair of complementary probes detected target DNA at a concentration of approximately 0.1 nM in 10 min or about 10 pM in 20-30 min. Detection of a 4 pM concentration of target DNA was demonstrated in 6 h using multiple probe pairs. The major limiting factors were background fluorescence and hybridization rates. Continuous monitoring of fluorescence during competitive hybridization allowed correction for variable sample backgrounds at probe concentrations down to 20 pM; however, the time required for complete hybridization increased to greater than 1 h at probe concentrations below 0.1 nM. A promising application for this technology is the rapid detection of amplified polynucleotides. Detection of 96,000 target DNA molecules in a 50-microliters sample was demonstrated following in vitro amplification using the polymerase chain reaction technique.