Immobilized DNA hairpins for assay of sequential breaking and joining of DNA backbones

Scott, Benjamin O. S., Lavesa-Curto, Manuel, Bullard, Desmond R., Butt, Julea N. and Bowater, Richard P. (2006) Immobilized DNA hairpins for assay of sequential breaking and joining of DNA backbones. Analytical Biochemistry, 358 (1). pp. 90-98. ISSN 1096-0309

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Abstract

Immobilized DNA hairpins are exploited in a novel approach to assay DNA ligases and nucleases. A fundamental characteristic of the assay is that a fluorophore at the remote terminus of the hairpin reports on the integrity of the DNA backbone. The functionality of the protocol is confirmed using ATP- and NAD+-dependent DNA ligases and the nicking enzyme N.BbvCIA. The assay format is amenable to high-throughput analysis and quantitation of enzyme activity, and it is shown to be in excellent agreement with the more laborious electrophoretic approaches that are widely used for such analyses. Significantly, the assay is used to demonstrate sequential breaking and rejoining of a specific nucleic acid. Thus, a simple platform for biochemically innovative studies of pathways in cellular nucleic acid metabolism is demonstrated.

Item Type: Article
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Rachel Smith
Date Deposited: 17 Feb 2011 15:04
Last Modified: 24 Sep 2019 16:30
URI: https://ueaeprints.uea.ac.uk/id/eprint/21408
DOI: 10.1016/j.ab.2006.08.010

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