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 trapping them in optical tweezers, is described in detail. Finally, we provide a protocol for carrying out optical tweezers experiments on the isolated mitotic chromosomes.
| Original language | English |
|---|---|
| Title of host publication | Single Molecule Analysis |
| Subtitle of host publication | Methods and Protocols |
| Editors | Iddo Heller, David Dulin, Erwin J.G. Peterman |
| Publisher | Humana Press Inc |
| Pages | 91-107 |
| Number of pages | 17 |
| Edition | 3rd |
| ISBN (Electronic) | 9781071633779 |
| ISBN (Print) | 9781071633793, 9781071633762 |
| DOIs | |
| Publication status | Published - 2024 |
Publication series
| Name | Methods in Molecular Biology |
|---|---|
| Volume | 2694 |
| ISSN (Print) | 1064-3745 |
| ISSN (Electronic) | 1940-6029 |
Bibliographical note
Publisher Copyright:© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024.
Funding
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.).
| Funders | Funder number |
|---|---|
| Horizon 2020 Framework Programme | 883240 |
| European Research Council |
Keywords
- Chromosome mechanics
- Force calibration for optical traps
- Microfluidics
- Mitotic chromosome isolation
- Mitotic chromosomes
- Optical tweezers