EdU induces DNA damage response and cell death in mESC in culture.

Recently, a novel DNA replication precursor analogue called 5-ethynyl-2'-deoxyuridine (EdU) has been widely used to monitor DNA synthesis as an alternative to bromodeoxyuridine. Use of EdU benefits from simplicity and reproducibility and the simple chemical detection systems allows excellent preservation of nuclear structure. However, the alkyne moiety is highly reactive, raising the possibility that incorporation might compromise genome stability. To assess the extent of possible DNA damage, we have analysed the effect of EdU incorporation into DNA during short- and long-term cell culture using a variety of cell lines. We show that EdU incorporation has no measurable impact on the rate of elongation of replication forks during synthesis. However, using different cell lines we find that during long-term cell culture variable responses to EdU incorporation are seen, which range from delayed cell cycle progression to complete cell cycle arrest. The most profound phenotypes were seen in mouse embryonic stem cells, which following incorporation of EdU accumulated in the G2/M-phase of the cell cycle before undergoing apoptosis. In long-term cell culture, EdU incorporation also triggered a DNA damage response in all cell types analysed. Our study shows that while EdU is extremely useful to tag sites of on-going replication, for long-term studies (i.e. beyond the cell cycle in which labelling is performed), a careful analysis of cell cycle perturbations must be performed in order to ensure that any conclusions made after EdU treatment are not a direct consequence of EdU-dependent activation of cell stress responses.

Visualising chromosomal replication sites and replicons in mammalian cells.

The precise regulation of DNA replication is fundamental to the preservation of intact genomes during cell proliferation. Our understanding of this process has been based traditionally on a combination of techniques including biochemistry, molecular biology and cell biology. In this report we describe how the analysis of the S phase in mammalian cells using classical cell biology techniques has contributed to our understanding of the replication process. We describe traditional and state-of-the-art protocols for imaging sites of DNA synthesis in nuclei and the organisation of active replicons along DNA, as visualised on individual DNA fibres. We evaluate how the different approaches inform our understanding of the replication process, placing particular emphasis on ways in which the higher order chromatin structures and the spatial architecture of replication sites contribute to the orderly activation of defined regions of the genome at precise times of S phase.

Successful mRNA profiling of 23 years old blood stains.

In this study, 23 years old human blood stains on different and problematic carrier materials, such as jeans, leather, wood, wallpaper, carpet, wool, and nylon fabric, were investigated by mRNA analysis of the blood markers HBB and SPTB. HBB turned out to be a very stable marker that could be detected in all blood samples from all carrier materials whereas SPTB showed no positive result in any of these old samples. Typing of co-extracted genomic DNA provided full STR profiles with the exception of one stain resulting in a partial profile. No differences were observed regarding the quality of results due to possible inhibitorial substances from carrier materials such as jeans or leather, whereas some difficulties to extract nucleic acids were caused by the physical properties of materials such as carpet fibres. HBB can be highly recommended for the identification of old blood stains in forensic casework as it was possible to detect HBB even in stains that failed to provide a full DNA profile.