Abstract
During mitosis, cells compact their DNA into rodlike shapes, four orders of magnitude shorter than the DNA backbone contour length. We describe an experimental protocol to isolate and study these intricate mitotic chromosomes using optical tweezers. We touch upon the technical details of the required optical tweezers and microfluidics setup, including advanced force calibration procedures to accurately measure the high forces the chromosomes withstand. The procedure used to isolate mitotic chromosomes, including biotinylation of the telomeric ends to facilitate trap** them in optical tweezers, is described in detail. Finally, we provide a protocol for carrying out optical tweezers experiments on the isolated mitotic chromosomes.
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Acknowledgments
We thank Anna E.C. Meijering, Christian F. Nielsen, Hannes Witt, Ian D. Hickson and Erwin J.G. Peterman for the development of the experimental workflow described here.
This work was supported by the European Research Council under the European Union’s Horizon 2020 research and innovation program (MONOCHROME, grant agreement no. 883240 to G.J.L.W.).
Competing Financial Interests
G.J.L.W. is a co-owner of LUMICKS B.V. The other authors declare no competing financial interest.
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Clement, T.V.M., van der Smagt, C., Wuite, G.J.L. (2024). Probing Mitotic Chromosome Mechanics Using Optical Tweezers. In: Heller, I., Dulin, D., Peterman, E.J. (eds) Single Molecule Analysis . Methods in Molecular Biology, vol 2694. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3377-9_5
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DOI: https://doi.org/10.1007/978-1-0716-3377-9_5
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Publisher Name: Humana, New York, NY
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