New applications of fluorescence polarization for enzyme assays and in genomics

Theo Nikiforov; Jill Coffin; Sang Jeong; Anton Simeonov; Xiahui Bi

doi:10.1117/12.426750

22 May 2001 New applications of fluorescence polarization for enzyme assays and in genomics

Theo Nikiforov, Jill Coffin, Sang Jeong, Anton Simeonov, Xiahui Bi

Proceedings Volume 4255, Clinical Diagnostic Systems; (2001) https://doi.org/10.1117/12.426750
Event: BiOS 2001 The International Symposium on Biomedical Optics, 2001, San Jose, CA, United States

Abstract

We have developed new, fluorescence polarization-based approaches for performing enzyme assays in homogeneous solutions and for detecting the hybridization of peptide nucleic acids to DNA targets. In the first method, fluorescein-labeled peptides serving as protein kinase sustrates are thiophosphorylated in the presence of the ATP analog ATPγS. A sulfer-reactive biotin derivative is then added to the mixture and allowed to react with the thiophosphorylated peptide. The formation of a fluorescein-labeled, biotinylated product can be detected by measuring the fluorescence polarization signal of fluorescein upon addition of streptavidin. In the second method, fluorescein-labeled peptides are subjected to enzymatic phosphorylation, desphosphorlation, or proteollytic cleavage by protein kinases, phosphatases, and proteases. The differential binding of the enzymatic substrates and products to cationic polymers such as polyaraginine can be conveniently measured by fluorescence polarization. Finally, we have discovered that the process of hybridization of peptide nucleic acid probes (PNAs) to their target DNA molecules can be followed by measuring the fluorescence polarization of a fluorophore attached to the PNA probes. These measurements can be done either in the presence or absence of polylysine in solution. Examples of the application of this method for single nucleotide polymorphism (SNP) typing are presented.

Citation Download Citation

Theo Nikiforov, Jill Coffin, Sang Jeong, Anton Simeonov, and Xiahui Bi "New applications of fluorescence polarization for enzyme assays and in genomics", Proc. SPIE 4255, Clinical Diagnostic Systems, (22 May 2001); https://doi.org/10.1117/12.426750

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