Chapter title |
Detection and Sequencing of Okazaki Fragments in S. cerevisiae.
|
---|---|
Chapter number | 10 |
Book title |
DNA Replication
|
Published in |
Methods in molecular biology, January 2015
|
DOI | 10.1007/978-1-4939-2596-4_10 |
Pubmed ID | |
Book ISBNs |
978-1-4939-2595-7, 978-1-4939-2596-4
|
Authors |
Smith, Duncan J, Yadav, Tejas, Whitehouse, Iestyn, Duncan J. Smith, Tejas Yadav, Iestyn Whitehouse, Smith, Duncan J. |
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. |
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Demographic breakdown
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Members of the public | 2 | 67% |
Scientists | 1 | 33% |
Mendeley readers
Geographical breakdown
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Unknown | 19 | 100% |
Demographic breakdown
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Student > Ph. D. Student | 8 | 42% |
Researcher | 3 | 16% |
Student > Master | 2 | 11% |
Student > Bachelor | 1 | 5% |
Student > Doctoral Student | 1 | 5% |
Other | 1 | 5% |
Unknown | 3 | 16% |
Readers by discipline | Count | As % |
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Agricultural and Biological Sciences | 5 | 26% |
Computer Science | 1 | 5% |
Engineering | 1 | 5% |
Unknown | 2 | 11% |