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 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.
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 also is 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 68.5°), 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 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 by which 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 (Fig. (Fig.1).1).
5′-nuclease assay with FP detection.
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 FP. We report here that both fluorescence intensity and FP gave completely concordant genotypes.
Some of TaqMan probes were designed for the antisense sequence (NCBI Assay ID ⧣231, 241, 686, 693, 3188, 3388, 2043, 3308, and 2619). The bases under the Allele 1 and Allele 2 columns were those reported in the dbSNP entries.
The results of the first 30 samples of the experiment are shown.
Acknowledgments
This work is supported in part by a grant from the National Institutes of Health (EY12557) to PYK. S.L. is a recipient of a Howard Hughes Medical Institute summer research fellowship. We thank S. Miller for informatics help.
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Footnotes
E-MAIL ude.ltsuw.sciteneg@kowk; FAX (314) 362-8159.
Article and publication are at www.genome.org/cgi/doi/10.1101/gr.156601