Int. J. Dev. Biol. 60: 297 - 304 (2016)

https://doi.org/10.1387/ijdb.160139oh

Vol 60, Issue 7-8-9

Special Issue: Cell-free Extracts in Development & Cancer Research

Single-molecule, antibody-free fluorescent visualisation of replication tracts along barcoded DNA molecules

Open Access | Technical Article | Published: 20 May 2016

Francesco De Carli^1,2, Vincent Gaggioli^1,3, Gaël A. Millot^4,5 and Olivier Hyrien*^,1

¹Institut de Biologie de l'Ecole Normale Supérieure (IBENS), CNRS UMR 8197, Inserm U1024, Ecole Normale Supérieure, PSL Research University, Paris, France, ²Sorbonne Universités, UPMC Univ Paris 06, IFD, Paris, France, ³Welcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, England, UK, ⁴Institut Curie, PSL Research University, CNRS, UMR 3244, Paris, France and ⁵Sorbonne Universités, UPMC Univ Paris 06, CNRS, UMR 3244, Paris, France.

Abstract

DNA combing is a standard technique to map DNA replication at the single molecule level. Typically, replicating DNA is metabolically labelled with nucleoside or nucleotide analogs, purified, stretched on coverslips and treated with fluorescent antibodies to reveal tracts of newly synthesized DNA. Fibres containing a locus of interest can then be identified by fluorescent in situ hybridization (FISH) with DNA probes. These steps are complex and the throughput is low. Here, we describe a simpler, antibody-free method to reveal replication tracts and identify the locus of origin of combed DNA replication intermediates. DNA was replicated in Xenopus egg extracts in the presence of a fluorescent dUTP. Purified DNA was barcoded by nicking with Nt.BspQI, a site-specific nicking endonuclease (NE), followed by limited nick-translation in the presence of another fluorescent dUTP. DNA was then stained with YOYO-1, a fluorescent DNA intercalator, and combed. Direct epifluorescence revealed the DNA molecules, their replication tracts and their Nt.BspQI sites in three distinct colours. Replication intermediates could thus be aligned to a reference genome map. In addition, replicated DNA segments showed a stronger YOYO-1 fluorescence than unreplicated segments. The entire length, replication tracts, and NE sites of combed DNA molecules can be simultaneously visualized in three distinct colours by standard epifluorescence microscopy, with no need for antibody staining and/or FISH detection. Furthermore, replication bubbles can be detected by quantitative YOYO-1 staining, eliminating the need for metabolic labelling. These results provide a starting point for genome-wide, single-molecule mapping of DNA replication in any organism.

Keywords

DNA replication, DNA combing, DNA barcoding, single-molecule analysis, Xenopus egg extract