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Methods
2012 Jun 01;572:179-86. doi: 10.1016/j.ymeth.2012.03.033.
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Single-molecule analysis of DNA replication in Xenopus egg extracts.
Yardimci H
,
Loveland AB
,
van Oijen AM
,
Walter JC
.
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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.
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