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P2074
High-Throughput Assays for de novo DNA Synthesis using Click Chemistry Detection Technology
Presenter Scott Clarke, Molecular Probes, USA
Additional Authors: Dani Hill, Iain Johnson, Yih-Tai Chen
Determination of cell proliferation through the quantitation of an incorporated nucleoside analog is the predominate method for directly measuring de novo DNA synthesis.  Direct measurement is useful for screening libraries of drugs for stimulation or inhibition of the S-phase of the cell cycle.  Here, a new assay is demonstrated where 5-ethynyl-2´-deoxyuridine (EdU) incorporation replaces BrdU in a HTS platform.  Detection and quantitation using click chemistry followed by fluorogenic Amplex® UltraRed substrate turnover is the basis of the assay.  Click chemistry which consists of a copper(I) catalyzed reaction forming a stable covalent bond between an azide and a terminal alkyne, offers a unique bio-orthogonal solution toward labeling nucleoside incorporated into DNA.  Cells are first treated with drug then pulsed with EdU, an alkyne nucleoside.  Simple fixation combined with a click reaction, covalently binds a detection tag to the DNA.  The use of click chemistry coupled with an enzymatic signal amplification replaces the traditional antibody-enzyme based amplification of BrdU signal.  Simple and rapid work flow with reduced number of washes, give the click based reaction an advantage in high-throughput screening.  Kinetic fluorescence determination or stable endpoint assays are readily adapted to large number of plates.  Examples of dose dependent IC50 curves generated from cell cycle inhibitors are compared to those derived from commercially available BrdU based assays.  HTS compatible advantages of reduced liquid handling and assay time, and stable signal read window are highlighted.