Journal
NATURE PROTOCOLS
Volume 5, Issue 7, Pages 1362-1377Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2010.102
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Funding
- Canadian Institutes of Health Research [RMF-92093]
- Michael Smith Foundation for Health Research
- Canadian Cancer Society Research Institute
- Terry Fox Foundation
- British Columbia Cancer Agency
- Canada Foundation for Innovation
- British Columbia Knowledge Development Fund
- British Columbia Cancer Foundation
- University of British Columbia
- NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES [R01GM094146] Funding Source: NIH RePORTER
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Previously, assays for sister chromatid segregation patterns relied on incorporation of 5-bromo-2'-deoxyuridine (BrdU) and indirect methods to infer segregation patterns after two cell divisions. In this study, we describe a method to differentially label sister chromatids of mouse cells and to directly assay sister chromatid segregation patterns after one cell division in vitro and in vivo by adaptation of the well-established COCO-FISH technique. BrdU is incorporated into newly formed DNA strands, which are then subjected to photolysis and exonuclease digestion to create single-stranded sister chromatids containing parental template DNA only. Such single-stranded sister chromatids are differentially labeled using unidirectional probes to major satellite sequences coupled to fluorescent markers. Differentially labeled sister chromatids in postmitotic cells are visualized using fluorescence microscopy, and sister chromatid segregation patterns can be directly assayed after one cell division. This procedure requires 4 d for in vivo mouse tissues and 2 d for in vitro-cultured cells.
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