Single-molecule analysis of DNA replication in Xenopus egg extracts

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Single-molecule analysis of DNA replication in Xenopus egg extracts

Yardimci, Hasan; Loveland, Anna B; van Oijen, Antoine M; Walter, Johannes C; (2012) Single-molecule analysis of DNA replication in Xenopus egg extracts. Methods , 57 (2) pp. 179-186. 10.1016/j.ymeth.2012.03.033. Green open access

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Abstract

The recent advent in single-molecule imaging and manipulation methods has made a significant impact on the understanding of molecular mechanisms underlying many essential cellular processes. Single-molecule techniques such as electron microscopy and DNA fiber assays have been employed to study the duplication of genome in eukaryotes. Here, we describe a single-molecule assay that allows replication of DNA attached to the functionalized surface of a microfluidic flow cell in a soluble Xenopus leavis egg extract replication system and subsequent visualization of replication products via fluorescence microscopy. We also explain a method for detection of replication proteins, through fluorescently labeled antibodies, on partially replicated DNA immobilized at both ends to the surface.

Type:	Article
Title:	Single-molecule analysis of DNA replication in Xenopus egg extracts
Location:	United States
Open access status:	An open access version is available from UCL Discovery
DOI:	10.1016/j.ymeth.2012.03.033
Publisher version:	https://doi.org/10.1016/j.ymeth.2012.03.033
Language:	English
Additional information:	This version is the author-accepted manuscript. For information on re-use, please refer to the publisher’s terms and conditions.
Keywords:	Single-molecule, DNA replication, Fluorescence microscopy
UCL classification:	UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Structural and Molecular Biology
URI:	https://discovery.ucl.ac.uk/id/eprint/10191056