Fluorescence Polarization in Homogeneous Nucleic Acid Analysis II: 5′-Nuclease Assay

When the temperature and viscosity of the solvent is held constant, the degree of fluorescence polarization (FP) detected when a fluorescent dye is excited by plane polarized light depends mostly on the molecular weight of the dye molecule. By monitoring the FP of a fluorescent dye molecule, one can detect significant changes in the molecular weight of a fluorescent molecule without separation or purification. The 5'-nuclease (TaqMan) assay is a robust single nucleotide polymorphism genotyping method where an allele-specific probe that binds to a perfectly complementary target is cleaved by the 5'-nuclease activity of Taq DNA polymerase. Because the TaqMan probe is labeled with a fluorescent dye, it has high FP value when intact but a low FP value after cleavage. In this study, we compared the results of the 5'-nuclease assay based on standard fluorescence intensity readings and FP readings when genotyping 90 individuals with 20 single nucleotide polymorphisms. Our results show that FP is just as robust and reliable as the standard fluorescence detection method. Use of FP detection makes it possible to reduce the cost of TaqMan probes by abrogating the need for a fluorescence quencher.