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Highly sensitive detection of DNA phosphorylation by counting single nanoparticles

TitleHighly sensitive detection of DNA phosphorylation by counting single nanoparticles
Publication TypeJournal Article
Year of Publication2010
AuthorsMa CB, Yeung ES
Journal TitleAnalytical and Bioanalytical Chemistry
Volume397
Pages2279-2284
Date Published07/01
ISBN Number1618-2642
Accession NumberISI:000279453000029
KeywordsAMPLIFICATION, DNA, enzyme, kinase activity, molecule detection, nucleic-acids, phosphorylation, PROTEIN MOLECULES, QUANTIFICATION, quantitation, single particle, strand break repair, t4 polynucleotide kinase, termini
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 x 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.
URL<Go to ISI>://000279453000029
DOIDoi 10.1007/S00216-010-3801-X