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Single-molecule analysis of DNA replication in Xenopus egg extracts

Journal Article


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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.

Authors


  •   Yardimci, Hasan (external author)
  •   Loveland, Anna B. (external author)
  •   van Oijen, Antoine M.
  •   Walter, Johannes C. (external author)

Publication Date


  • 2012

Citation


  • Yardimci, H., Loveland, A. B., van Oijen, A. M. & Walter, J. C. (2012). Single-molecule analysis of DNA replication in Xenopus egg extracts. Methods, 57 (2), 179-186.

Scopus Eid


  • 2-s2.0-84865560133

Ro Full-text Url


  • http://ro.uow.edu.au/cgi/viewcontent.cgi?article=3188&context=smhpapers

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers/2170

Has Global Citation Frequency


Number Of Pages


  • 7

Start Page


  • 179

End Page


  • 186

Volume


  • 57

Issue


  • 2

Place Of Publication


  • United States

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.

Authors


  •   Yardimci, Hasan (external author)
  •   Loveland, Anna B. (external author)
  •   van Oijen, Antoine M.
  •   Walter, Johannes C. (external author)

Publication Date


  • 2012

Citation


  • Yardimci, H., Loveland, A. B., van Oijen, A. M. & Walter, J. C. (2012). Single-molecule analysis of DNA replication in Xenopus egg extracts. Methods, 57 (2), 179-186.

Scopus Eid


  • 2-s2.0-84865560133

Ro Full-text Url


  • http://ro.uow.edu.au/cgi/viewcontent.cgi?article=3188&context=smhpapers

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers/2170

Has Global Citation Frequency


Number Of Pages


  • 7

Start Page


  • 179

End Page


  • 186

Volume


  • 57

Issue


  • 2

Place Of Publication


  • United States