The genetic mutations driving the apparition of tumors are commonly linked to environmental and intrinsic factors. The part of environmental risk versus mistakes in DNA replication have been heavily discuted through the year. In 2015, a publication by Vogelstein and Thomasetti bring a new theory explaining why some cancers occur more than others (Tomasetti and Vogelstein, 2015). Using statistical analysis of cancer cases in the U.S., they concluded that differences in the number of stem cell divisions in an organ are strongly correlated (0.81) with the total number of divisions of stem cells maintaining that tissue’s homeostasis with the frequency of cancers in that area. There aren’t as many stem cells in the brain, where few cancers occurred, than in the colon and intestine where occurred a lot of colorectal cancer. As this study is very controversial, hundred of studies have been written in response, bringing this topic update. Last paper of Vogelstein and Thomasetti conclude that the role of DNA typos is organ related, and indicated that about 66% of cancer-driving mutations are due to random DNA replication errors, with only 29% due to environmental factors and 5% to inherited mutations (Tomasetti et al., 2017).

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Despite continuous improvement of DNA FISH, a method that has been extensively used for years, it still requires harsh treatments to allow probe hybridization. Oligoprobes are also expansive, and the method is time consuming. Teams are thus looking for improvements. For this purpose, Deng et al. from the Transcription Imaging Consortium (Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147) have used the CRISPR/Cas9 system to allow highly specific and efficient labeling of DNA without global DNA denaturation, which is generated by heat or chemical treatments in DNA FISH protocols.

Let’s quote the significance they attach to their work:

We have derived a new technology for the detection of genes within undisturbed nuclei of fixed cells and tissues. Previous approaches have used fluorescent DNA probes to hybridize to genes of interest, requiring treatment of heat and disruptive chemicals that distort the natural organization of the nucleus. Instead, we have used a bacterial protein, CRISPR (clustered regularly interspaced short palindromic repeats), combined with an RNA sequence as probes to find the genes of interest in the intact genome. This approach preserves the spatial relationships of the genetic elements, which are important for understanding gene expression, and the process is remarkably rapid (15 min), convenient, and can be used directly on tissues for diagnosis of disease.

This article is available here, and an explained abstract can be read on the very good “greenfluorescentblog” !

The comet assay is a sensitive, well established technique for quantifying DNA damage in eukaryotic cells. Compatible with the detection of a wide range of DNA damaging agents, its principle consists in the migration of fragmented DNA in an electrophoresis gel (damaged DNA forming the tail of the comet), while intact DNA moves at a slower rate (head of the comet). The percentage of fragmented DNA in the comet tail is a direct measure of DNA damage.

The comets are evaluated via fluorescence staining and microscopy. Two drawbacks are often mentioned about the comet assay: preparing the comet slides with cells in agarose to perform electrophoresis is time consuming and reduce the throughput of the method, and, if manual scoring of the comets are done, there can be a high inter-operator variability.

In HCS Pharma, We have combined processes to perform an automated comet assay, from the cell and agarose handling, to comet assessment and scoring, by using automated instrumentation and a 96-well format comet slide. This fully automated process can allow us to test your compounds or active ingredients in medium throughut (96-well plates) either in genotoxicity assays or in efficacy assay as DNA damage protection assays .

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