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Methods. 2012 Jun;57(2):179-86. doi: 10.1016/j.ymeth.2012.03.033. Epub 2012 Apr 6.

Single-molecule analysis of DNA replication in Xenopus egg extracts.

Author information

1
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.

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.

PMID:
22503776
PMCID:
PMC3427465
DOI:
10.1016/j.ymeth.2012.03.033
[Indexed for MEDLINE]
Free PMC Article

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