Methods Mol Biol 2009 Jan 01;521:575-603. doi: 10.1007/978-1-60327-815-7_33.

Use of DNA combing to study DNA replication in Xenopus and human cell-free systems.

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The Xenopus egg extract has become the gold standard for in vitro studies of metazoan DNA replication. We have used this system to study the mechanisms that ensure rapid and complete DNA replication despite random initiation during Xenopus early development. To this end we adapted the DNA combing technique to investigate the distribution of replication bubbles along single DNA molecules. DNA replicating in egg extracts is labelled by addition of digoxigenin-11-dUTP and/or biotin-16-dUTP at precise times. These two dTTP analogues are efficiently incorporated into DNA during replication in the extract. After DNA purification and combing the DNA is visualized with appropriate fluorescent antibody/streptavidin molecules. Replicated DNA appears as green or red tracts whose pattern reveals how each molecule was replicated, allowing to follow the dynamics of DNA replication through S phase. We describe (a) the preparation and use of egg extracts and demembranated sperm chromatin templates; (b) a simple method for preparing silanized glass coverslips suitable for DNA combing and fluorescence detection; (c) two alternative replicative DNA labelling schemes and their respective advantages; and (d) a protocol for combining replicative labelling with detection of specific DNA sequences by fluorescent in situ hybridization (FISH). Although most observations made in Xenopus egg extracts are applicable to other eukaryotes, there are differences in cell-cycle regulation between mammalian somatic cells and embryonic amphibian cells, which led to the development of human cell-free systems that can initiate semi-conservative chromosomal DNA replication under cell-cycle control. We have employed the knowledge gained with Xenopus extracts to characterize DNA replication intermediates generated in human cell-free systems using DNA combing. We describe here (a) the preparation and use of human cell-free extracts and initiation-competent template nuclei for DNA combing studies; (b) an optimized labelling scheme for DNA replication intermediates by molecular combing and fluorescence microscopy.

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