Genome

Methods. 2012 Jul 11. [Epub ahead of print]

Genome-scale identification of active DNA replication origins.

Cayrou C, Grégoire D, Coulombe P, Danis E, Méchali M.

Source

Institute of Human Genetics, CNRS, 141 Rue de la Cardonille, 34396 Montpellier, France.

Abstract

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.

Copyright ? 2012. Published by Elsevier Inc.