BioSpotlight

• Published in: BioTechniques Vol. 55; no. 4; p. 159
• Main Authors: Lo, Patrick C. H, Nybo, Kristie
• Format: Journal Article
• Language: English
• Published: Future Science Ltd 01.10.2013; Taylor & Francis Group
• ISSN: 0736-6205; 1940-9818
• DOI: 10.2144/000114082

Analyzing the dynamics of replication fork progression is critical for understanding DNA replication and repair as well as epigenetic regulation involving the deposition of histones and other chromatin proteins. Recently, isolation of protein on nascent DNA (iPOND) was developed to purify proteins found at the replication fork. Nascent DNA at the replication fork is labeled through the incorporation of a brief pulse of the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU), followed by formaldehyde crosslinking of chromatin proteins to DNA and the covalent linkage of biotin azide to the alkyne group of EdU through a copper-catalyzed "click" reaction. After sonication of the chromatin, biotinylated nascent DNA and associated proteins are isolated using streptavidin-coated beads and proteins are analyzed by Western blotting after the reversal of the formaldehyde crosslinking. iPOND, however, has drawbacks, including modest protein yields and the need for formaldehyde crosslinking that can affect Western blotting and mass spectrometry protein analysis.