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

Benjamin O. S. Scott; Manuel Lavesa-Curto; Desmond R. Bullard; Julea N. Butt; Richard P. Bowater

doi:10.1016/j.ab.2006.08.010

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

Benjamin O. S. Scott, Manuel Lavesa-Curto, Desmond R. Bullard, Julea N. Butt, Richard P. Bowater

Research output: Contribution to journal › Article › peer-review

7 Citations (Scopus)

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.

Original language	English
Pages (from-to)	90-98
Number of pages	9
Journal	Analytical Biochemistry
Volume	358
Issue number	1
DOIs	https://doi.org/10.1016/j.ab.2006.08.010
Publication status	Published - 1 Nov 2006

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10.1016/j.ab.2006.08.010

Cite this

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title = "Immobilized DNA hairpins for assay of sequential breaking and joining of DNA backbones",

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.",

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AU - Scott, Benjamin O. S.

AU - Lavesa-Curto, Manuel

AU - Bullard, Desmond R.

AU - Butt, Julea N.

AU - Bowater, Richard P.

PY - 2006/11/1

Y1 - 2006/11/1

N2 - 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.

AB - 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.

U2 - 10.1016/j.ab.2006.08.010

DO - 10.1016/j.ab.2006.08.010

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VL - 358

SP - 90

EP - 98

JO - Analytical Biochemistry

JF - Analytical Biochemistry

SN - 0003-2697

IS - 1

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