Detection and sequencing of okazaki fragments in S. cerevisiae Journal Article


Authors: Smith, D. J.; Yadav, T.; Whitehouse, I.
Article Title: Detection and sequencing of okazaki fragments in S. cerevisiae
Abstract: We have previously demonstrated that lagging-strand synthesis in budding yeast is coupled with chromatin assembly on newly synthesized DNA. Using a strain of S. cerevisiae in which DNA ligase I can be conditionally depleted, we can enrich and purify Okazaki fragments. We delineate a method to extract, end label, and visualize Okazaki fragments using denaturing agarose gel electrophoresis. Furthermore, we describe an ion-exchange chromatographic method for purification of fragments and preparation of strand-specific sequencing libraries. Deep sequencing of Okazaki fragments generates a comprehensive, genomic map of DNA synthesis, starting from a single asynchronous culture. Altogether this approach represents a tractable system to investigate key aspects of DNA replication and chromatin assembly. © 2015, Springer Science+Business Media New York.
Keywords: genetics; nonhuman; dna replication; dna synthesis; animal cell; metabolism; gene mapping; dna; saccharomyces cerevisiae; chromatin; staining; dna sequence; saccharomycetales; okazaki fragments; okazaki fragment; chromatin assembly and disassembly; labeling index; staining and labeling; sequence analysis, dna; dna, fungal; genome, fungal; budding yeast; dna purification; agarose; agar gel electrophoresis; fungal strain; fungal dna; fungal genome; fungus culture; procedures; lagging strand; dna library; priority journal; article; electrophoresis, agar gel; denaturing agarose gel electrophoresis
Journal Title: Methods in Molecular Biology
Volume: 1300
ISSN: 1064-3745
Publisher: Humana Press Inc  
Date Published: 2015-01-01
Start Page: 141
End Page: 153
Language: English
DOI: 10.1007/978-1-4939-2596-4_10
PUBMED: 25916711
PROVIDER: scopus
PMCID: PMC4860728
DOI/URL:
Notes: Chapter 10 in "DNA Replication" (ISBN: 978-1-4939-2595-7) -- Export Date: 21 June 2016 -- Source: Scopus
Altmetric
Citation Impact
BMJ Impact Analytics
MSK Authors
  1. Duncan John Smith
    5 Smith