Journal
METHODS
Volume 57, Issue 2, Pages 179-186Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymeth.2012.03.033
Keywords
Single-molecule; DNA replication; Fluorescence microscopy
Funding
- NIH [GM077248, GM62267, GM80676]
- ACS [RSG0823401GMC]
- Netherlands Organization for Scientific Research (NWO) [Vici 680-47-607]
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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. (c) 2012 Elsevier Inc. All rights reserved.
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