期刊
METHODS
卷 57, 期 2, 页码 158-164出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymeth.2012.06.015
关键词
DNA replication origins; Nascent strand purification; Lambda-exonuclease; Microarrays
资金
- European Research Council (ERC, FP7 Grant) [233339]
- Agence Nationale de la Recherche (ANR)
- Association pour la Recherche sur le Cancer (ARC)
- 'Ligue Nationale Contre le Cancer' (LNCC)
- European Research Council (ERC) [233339] Funding Source: European Research Council (ERC)
Understanding the nature of DNA replication origins in metazoan is quite challenging. In the absence of a genetic assay like in yeast, methods were devised based on the DNA structure, the visualization or quantification of the first nascent strands that are synthesized at origins, or on the localization of origin binding proteins. The purification and quantification of RNA-primed nascent DNA at origins during initiation of DNA synthesis is the most exhaustive and precise method to map active replication origins at present. We have upgraded this method to the level of reproducibility and enrichment required for genome-wide analyses by microarrays or deep sequencing. We detail here the protocol and the controls required at the different steps. (c) 2012 Published by Elsevier Inc.
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