Single-molecule Analysis of DNA Replication Dynamics in Budding Yeast and Human Cells by DNA Combing
The DNA combing method allows the analysis of DNA replication at the level of individual DNA molecules stretched along silane-coated glass coverslips. Before DNA extraction, ongoing DNA synthesis is labeled with halogenated analogues of thymidine. Replication tracks are visualized by immunofluoresce...
| Published in: | Bio-Protocol |
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| Main Authors: | , , , |
| Format: | Article |
| Language: | English |
| Published: |
Bio-protocol LLC
2017-06-01
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| Online Access: | https://bio-protocol.org/en/bpdetail?id=2305&type=0 |
| Summary: | The DNA combing method allows the analysis of DNA replication at the level of individual DNA molecules stretched along silane-coated glass coverslips. Before DNA extraction, ongoing DNA synthesis is labeled with halogenated analogues of thymidine. Replication tracks are visualized by immunofluorescence using specific antibodies. Unlike biochemical and NGS-based methods, DNA combing provides unique information on cell-to-cell variations in DNA replication profiles, including initiation and elongation. Finally, this assay can be used to monitor the effect of DNA lesions on fork progression, arrest and restart. |
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| ISSN: | 2331-8325 |