Detection and sequencing of okazaki fragments in S. cerevisiae

Duncan J. Smith, Tejas Yadav, Iestyn Whitehouse

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)141-153
Number of pages13
JournalMethods in Molecular Biology
Volume1300
DOIs
StatePublished - 2015

Keywords

  • Budding yeast
  • Chromatin
  • DNA replication
  • Lagging strand
  • Okazaki fragments

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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