Creating localized DNA double-strand breaks with microirradiation.
Nat Protoc
; 6(2): 134-9, 2011 Feb.
Article
em En
| MEDLINE
| ID: mdl-21293454
ABSTRACT
We describe a protocol for creating localized DNA double-strand breaks (DSBs) with minimal requirements that can be applied in cell biology and molecular biology. This protocol is based on the combination of 5-bromo-2'-deoxyuridine (BrdU) labeling and ultraviolet C (UVC) irradiation through porous membranes. Cells are labeled with 10 µM BrdU for 48-72 h, washed with Ca(2+)- and Mg(2+)-free PBS(-), covered by polycarbonate membranes with micropores and exposed to UVC light. With this protocol, localized DSBs are created within subnuclear areas, irrespective of the cell cycle phase. Recruitment of proteins involved in DNA repair, DNA damage response, chromatin remodeling and histone modifications can be visualized without any specialized equipment. The quality is the same as that obtained by laser microirradiation or by any other focal irradiation. DSBs become visible within 30 min of UVC irradiation.
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1
Base de dados:
MEDLINE
Assunto principal:
Mutagênese Sítio-Dirigida
/
Quebras de DNA de Cadeia Dupla
Idioma:
En
Ano de publicação:
2011
Tipo de documento:
Article