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 |
