title: Single-molecule analysis of DNA replication in Xenopus egg extracts.
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.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.
authors.forename: Hasan
authors.initials: H
authors.lastname: Yardimci
authors.forename: Anna B
authors.initials: AB
authors.lastname: Loveland
authors.forename: Antoine M
authors.initials: AM
authors.lastname: van Oijen
authors.forename: Johannes C
authors.initials: JC
authors.lastname: Walter
mesh_terms: D000818:Animals; D001483:Base Sequence; D002457:Cell Extracts / Q000235:genetics*; D002469:Cell Separation; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis* / Q000737:chemistry; D005260:Female; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D061925:Immobilized Nucleic Acids / Q000096:biosynthesis* / Q000737:chemistry; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation; D009865:Oocytes / Q000166:cytology*; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry; D011485:Protein Binding; D013194:Staining and Labeling; D014982:Xenopus laevis
chemical_list: D002457:Cell Extracts; D004279:DNA, Viral; D061925:Immobilized Nucleic Acids; D018809:Proliferating Cell Nuclear Antigen