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

Abstract

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 5Ј-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 5Ј-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 5Ј-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.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 allelespecific 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 also is a good detection method for the 5Ј-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 planepolarized 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 68.5°), 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 t...

SNP genotyping with fluorescence polarization detection

“…For the heterozygotes, both probes are cleaved and they occupy the origin of the plot, where both VIC and FAM FP values are low. The genotypes called by FP analysis were in complete concordance with those called by the conventional analysis method [Latif et al, 2001].…”

“…We have shown recently that FP detection is indeed suitable for the 5′-nuclease assay [Latif et al, 2001]. Twenty markers, previously optimized for the 5′-nuclease assay, were used in the study.…”

“…We have shown previously that fluorescence polarization (FP) is a good detection method in assays where the molecular weight of the starting fluorescent reagent changes significantly when converted into products. Indeed, FP detection has been shown to work well in the primer extension assay [Chen et al, 1999;Hsu et al, 2001b], in the 5′-nuclease (TaqMan ® ) assay [Latif et al, 2001], and in the Invader ® assay [Hsu et al, 2001a]. In this article, the use of FP detection and the three assays for which FP detection works well are described.…”

Induction of Heat Shock Proteins by Hyperthermia and Noise Overstimulation in Hsf1 −/− Mice

“…The difference in C T between the gene of interest (gene "X") and S16 for any given RNA sample was defined as ΔC T (X). The difference in ΔC T (X) between two samples (e.g., induced vs. uninduced control or noise-exposed vs. unexposed control) was defined as ΔΔC T (X), which represented a relative difference in expression of gene X (Latif et al 2001). Fold change was calculated after adjusting for amplification efficiency of the PCR assay for the corresponding gene.…”

Single nucleotide polymorphism genotyping: biochemistry, protocol, cost and throughput

“…The FP principle has been successfully applied to SBE, 35 DNA hybridization, 58,73 the Invader assay 61 and the TaqMan 5 0 -nuclease assay. 74 …”