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Highly sensitive detection of DNA phosphorylation by counting single nanoparticles

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Abstract

DNA phosphorylation is a vital process in the repair, replication, and recombination of nucleic acids. Traditionally, it is assayed by denaturing gel electrophoresis and autoradiography, which are tedious and not sensitive. We report on the development of a sensitive, simple, and economical method for DNA phosphorylation detection and T4 polynucleotide kinase (T4 PNK) activity assay based on marking DNA phosphorylation/biotinylation events by the attachment of fluorescent nanoparticles. Enzyme activity of T4 PNK is measured down to a limit of 5 × 10−6 U/ml, which is 400 times lower than previous reports. We also studied DNA phosphorylation specificity with different DNA substrates. Furthermore, T4 PNK inhibition by the inhibitor ADP and activation by the activator spermine are shown, demonstrating the potential for high-throughput screening for inhibitors and activators.

Sensitive detection of DNA phosphorylation is achieved by counting single nanoparticles, providing direct measurement of T4 PNK down to 5 x 10-6 U/mL, or 400 times lower than previous reports.

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Acknowledgments

The Ames Laboratory is operated for the U.S. Department of Energy by Iowa State University under Contract No. DE-AC02-07CH11358. This work was supported by the Director of Science, Office of Basic Energy Science, Division of Chemical Sciences.

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Authors and Affiliations

  1. Ames Laboratory-U.S. DOE and Department of Chemistry, Iowa State University, Ames, IA, 50011, USA

    Changbei Ma & Edward S. Yeung

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  1. Changbei Ma
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  2. Edward S. Yeung
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Correspondence to Edward S. Yeung.

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Ma, C., Yeung, E.S. Highly sensitive detection of DNA phosphorylation by counting single nanoparticles. Anal Bioanal Chem 397, 2279–2284 (2010). https://doi.org/10.1007/s00216-010-3801-x

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