FLUORESCENCE POLARIZATION IN HOMOGENEOUS NUCLEIC ACID ANALYSIS II: 5'-NUCLEASE ASSAY. Sherif Latif1, Irma Bauer-Sardina2, Kenneth J. Livak3, Pui-Yan Kwok2. Biology Department, Washington University, St. Louis, MO1; Division of Dermatology, Washington University Medical School, St. Louis, MO2; Applied Biosystems, Foster City, CA3.

The ability to determine efficiently and unambiguously the mutational status or genotype of an organism has great applications in molecular diagnostics, clinical genetic testing, population genetics, and agricultural biotechnology. High-throughput genotyping methods for single nucleotide variations currently in use discriminate between the alleles by differential hybridization, primer extension, ligation, and allele specific cleavage of a probe (Kwok 2000). Homogeneous assays based on these allele discrimination mechanisms are conducted in aqueous solutions without separation or purification by monitoring physical changes when the reagents are turned into products. We have shown previously that fluorescence polarization (FP) is a good detection method in the primer extension assay when a dye-labeled dideoxy terminator is incorporated allele-specifically in the presence of a matching DNA template (Chen et al., 1999). In this report, we show that FP is also a good detection method for the 5'-nuclease (TaqMan) assay, where a fluorescent probe is cleaved during the polymerase chain reaction only when it is annealed to a perfectly complementary template.

When a fluorescent molecule is excited by plane-polarized light at the correct wavelength, the fluorescence emitted is also polarized. However, because the molecule rotates and tumbles in space, the FP observed is proportional to the fluorescent molecule's rotational relaxation time (the time it takes to rotate through an angle of 68.5o), which is related to the viscosity of the solvent, absolute temperature, molecular volume, and the gas constant. Therefore, if the viscosity and temperature are constant, FP is directly proportional to the molecular volume, which is directly proportional to molecular weight. If the fluorescent molecule is large (with high molecular weight), it rotates and tumbles more slowly in space and FP is preserved. If the molecule is small (with low molecular weight), it rotates and tumbles faster and FP is largely lost or depolarized. In principle, FP can be used to detect any significant change in molecular weight of a fluorescent molecule. Indeed, FP detection is the basis of numerous clinical and research assays, especially those involved in ligand-receptor binding (Checovich et al. 1995).

The 5'-nuclease assay is one of the simplest diagnostic assays where one can determine the mutational status of a DNA sample in one step (Livak, 1999). The current detection method in the 5-nuclease assay relies on the increase in fluorescence intensity when a reporter fluorophore is released from its quencher as the doubly labeled probe is cleaved during PCR (Lee et al. 1999). Because the 5'-nuclease assay is one where a large probe is cleaved into small molecules, we reason that FP can be a good detection system for the method (see Figure 1).

To show that FP detection is indeed suitable for the 5-nuclease assay, we performed the assay with 20 markers on DNA samples from 90 individuals. At the end of the assay, detection and analysis was done by both fluorescence intensity and fluorescence polarization. We were able to report that both fluorescence intensity and fluorescence polarization gave completely concordant genotypes