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Single molecule sequencing and biophysical properties of oxidized genomic DNA using magnetic tweezers.

Project description

Scissors and tweezers may soon lead the pack of advanced DNA sequencing methods

DNA undergoes numerous biochemical and biophysical changes to modulate gene expression. These epigenetic modifications do not alter the DNA sequence but can affect how it is 'read'. A common example is methylation (addition of CH3), which physically impedes transcription and gene expression. While synthetic chemistry and DNA sequencing techniques have advanced tremendously, they do not preserve critically valuable epigenetic information. The EU-funded EpiSeq project is using high-tech 'molecular scissors' (CRISPR/Cas9 technology) to cut out the small sequence of DNA of interest, and magnetic tweezers to manipulate the molecule and determine its epigenetic sequence. Knowledge of epigenetic changes will inform studies on how gene expression changes cause certain diseases with implications for better diagnoses and treatment.

Objective

Next-generation DNA sequencing technologies are at the core of modern molecular biology and are rapidly entering the technological arsenal for personalized medicine. The recent explosion of sequencing technologies has provided researchers with various solutions to fit their sequencing needs, with a large spectrum of costs and limitations. However, most sequencing methods use enzymatic duplication of DNA to generate a strong enough sequencing signal. Therefore, chemical modifications potentially present on native bases are lost, and with them, an entire level of epigenetic information about. Although some workarounds are emerging, they are indirect, costly, and not yet amenable for the targeted sequencing of a given native genomic locus or biophysical analysis of the modified DNA. Given the need to understand the epigenetic layer of genetic information, I propose to develop a reliable and cheap method to map, at the single-molecule resolution, the epigenetic status of a genomic DNA locus of interest. This will be achieved through biochemical capture of this locus using CRISPR/Cas9 technology, and its epigenetic sequencing via single-molecule manipulation by magnetic tweezers. In addition to identifying the nature and the position of the modified bases at a given genomic DNA locus, I will use this magnetic tweezer technology to address fundamental questions about the effect of epigenetic modifications on the biophysical properties of DNA (formation of DNA structures and protein interactions). Specifically, I will focus on 8-oxoguanine, for which there is increasing interest since it is a hallmark of degenerative pathologies like cancer or Alzheimer’s Disease, and since it has recently entered the known epigenetic arsenal. This project has great potential applications in medical diagnosis. It will give me a first-hand experience in developing a cutting-edge technology, which will be of great benefit to my future independent research career.

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MSCA-IF-EF-RI - RI – Reintegration panel

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Call for proposal

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(opens in new window) H2020-MSCA-IF-2018

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Coordinator

MUSEUM NATIONAL D'HISTOIRE NATURELLE
Net EU contribution

Net EU financial contribution. The sum of money that the participant receives, deducted by the EU contribution to its linked third party. It considers the distribution of the EU financial contribution between direct beneficiaries of the project and other types of participants, like third-party participants.

€ 184 707,84
Address
RUE CUVIER 57
75005 Paris
France

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Region
Ile-de-France Ile-de-France Paris
Activity type
Higher or Secondary Education Establishments
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Total cost

The total costs incurred by this organisation to participate in the project, including direct and indirect costs. This amount is a subset of the overall project budget.

€ 184 707,84
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