Application of Fluorescence Polarization to Enzyme Assays and Single Nucleotide Polymorphism Genotyping: Some Recent Developments

Theo   T.   Nikiforov; Anton   M.   Simeonov

Abstract

This article describes some recent developments in the field of fluorescence polarization (FP) as applied to enzyme assays and single nucleotide polymorphism (SNP) genotyping. First, we present our recent progress on the application of fluorescence polarization to high throughput screening (HTS). We show how the use of a 2-thiopyridinone-based, mixed disulfide biotinylation reagent can shorten the assay time of our recently reported kinase assay method based on thiophosphorylation and biotinylation from several hours to a few minutes. We also summarize our recent findings on a new approach for HTS of kinases, proteases and phosphatases based on the use of a cationic poly-amino acid such as polyarginine. We show how the careful selection of the polyarginine concentration and the ionic strength of the solution during the FP measurement allow one to expand the range of substrates that can be assayed. Both of these methods are valuable additions to the existing techniques for HTS. Most importantly, both of these methods can be applied to the assay of kinases without the need for any antibodies. In the area of genomics, we present some results from our studies on a new single nucleotide polymorphism typing approach based on the polymerase catalyzed extension of 3 fluorescein labeled primers. Contrary to our initial expectations, we observed that the enzymatic extension of these primers results in a significant decrease of the fluorescence polarization value. Possible explanations of this phenomenon are discussed.

Keywords: Fluorescence polarization,, enzyme assays,, thiophosphorylation,, high throughput screening,, SNP genotyping,, polymerase extension.

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