The FP-TDI protocol was originally reported by Drs. Chen, Levine, and Kwok at Washington University in 1999^1,2. FP-TDI stands for template directed dye terminator incorporation assay with detection by fluorescence polarization. It is a single base primer extension assay couple with homogeneous FP detection.

• Assay Principle and Procedure		• Typical Data
• Fluorescence Polarization		• Advantages of FP-TDI
• Publications on FP-TDI

Assay Principle and Procedure

There are four key steps to a FP-TDI assay.

• Template Amplification by PCR protocol
• PCR product clean-up by Exo-SAP-It reagent
• Single-base primer extension: using a primer that anneals one base shy of the polymorphic site, and fluophore labeled terminators
• FP reading and data analysis

See below for an illustration.  (Click here for an animated version of the illustration)

For detailed step by step assay procedures, please click here to view the AcycloPrime Manual.

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Data from an analysis using DNA from separate individuals for a particular SNP tends to show to varying degrees that the data groups into 4 clusters. The lower left grouping is generally composed of experimental control blanks and samples with failed PCR reactions. The lower right grouping is from individuals homozygote for the one allele (the terminator containing the R110 dye). The upper left is from individuals homozygote for the other allele (the terminator containing the TAMRA dye). The last grouping in the upper right is from heterozygote individuals (labels with both R110 and TAMRA terminators). Several of the points may not clearly group with any cluster and may either be reanalyzed experimentally or use a of more sophisticated software analyses may permit calling of the SNP in these limited cases.

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Fluorescence Polarization

FP is a popular technique designed for homogeneous, high throughput assays. The principle behind the technique is molecular weight differences between two fluorescence labeled molecules. A small molecule containing a fluorophore spins around rapidly, and a large molecule containing a fluorophore spins around slowly. When a polarizing light is used to excite these fluorophores, small molecules will emit light in all directions, while large molecules will emit light only in the same plane, thus producing higher polarized fluorescence.

(Click here for an animated version of the illustration below)

The amount of fluorescence polarization as measured by mP value is directly correlated to molecular weight in a dynamic range from 1KD (one nucleotide base) to about 10KD (a 30mer oligonucleotide).

Learn More........

For more information on FP, refer to article by J. Owicki.

John C. Owicki. J. Biomolecular Screening 5(5), 287, 2000 Link: http://www.liebertpub.com/BSC/default1.asp

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Quoting from Dr. Pui-Yan Kwok's article in

Biotechniques, "Although the FP-TDI assay is one of several different methods that are currently available for automated genotyping, it has a number of advantages."

• This assay is less costly because it does not require any labeled primer. Reagent cost is generally around $0.50 per SNP or less. Instruments for FP reading costs around $30,000 at the low end.
• Single-base incorporation assays are very easy to optimize. One can literally develop a robust assay within hours of obtaining the primers. No redesigning or lengthy manufacturing of specialty probes or microarrays is necessary when a new marker is needed for a study.
• FP-TDI assay uses FP as a detection format, which is independent of fluorescence intensity and requires no separation of free from bound dye ddNTP.
• FP-TDI can be adopted at low volume SNP labs with just a FP reader, or scaled up to high throughput screening with full automation, delivering over 50,000 genotypings per day.
• Complete FP-TDI solution (instruments, reagents, software) can be obtained from one source: PerkinElmer Life & Analytical Sciences.

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Publications on FP-TDI

1. Chen, X., Levine, L. and Kwok, P-Y., “Fluorescence polarization in homogeneous nucleic acid analysis”, Genome Res. (1999) 9,492-498

2. Hsu, T.M., Chen, X., Duan, S., Miller, R.D., and Kwok, P-Y., “Universal SNP Genotyping assay with fluorescence polarization detection”, Biotechniques (2001) 31:560-570.

3. Hamilton, S., Slager, S., Heiman, G., Deng, Z., Haghighi, F., Klein, D., Hodge, S, Weissman, M., Fyer, A., Knowles, J. “Evidence for a susceptibility Locus for Panic Disorder near the Catechol-O-Methyltransferase Gene on Chromosome 22”, Biol. Psychiatry (2002) 51:591-601.

4. Akula, N., Chen, YS., Hennessay, K., Schulze, TG., Singh, G., and McMahon, FJ. "Utility and Accuracy of Template-Directed Dye-Terminator Incorporation with Fluorescence Polarization Detection for Genotyping Single Nucleotide Polymorphisms", Biotechniques (2002) 32:1072-1079.

5. Kathryn A. Swan, Damian E. Curtis, Kathleen B. McKusick, Alexander V. Voinov, Felipa A. Mapa, and Michael R. Cancilla "High-Throughput Gene Mapping in Caenorhabditis elegans", Genome Research (2002): 12: 1100-1105

6. Kwok, Pui-Yan. “SNP Genotyping with fluorescence Polarization Detection”, Human Mutation (2002) 19: 315-323.

7. Richard A. Greene, James J. DiMeo, Mary E. Malone, Suzanne Swartwout, Jianzhao Liu and Philip R. Buzby. "A Novel Method for SNP Analysis Using Fluorescence Polarization", PerkinElmer Life Sciences, P10131 rev. 07/01.

8. Freeman BD, Buchman TG, McGrath S, Tabrizi AR and Zehnbauer BA. "Template-Directed Dye-Terminator Incorporation with Fluorescence Polarization Detection for Analysis of Single Nucleotide Polymorphisms Implicated in Sepsis", Journal of Molecular Diagnostics (2002) : Vol. 4, No. 4 : 209 - 215.

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