In situ detection of specific DNA double strand breaks using rolling circle amplification.

Imagen de Paul Lizardi
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TítuloIn situ detection of specific DNA double strand breaks using rolling circle amplification.
Publication TypeJournal Article
Year of Publication2005
AutoresLi, J, Young, CSH, Lizardi, PM, Stern, DF
JournalCell Cycle
Volume4
Issue12
Pagination1767-73
Date Published2005 Dec
ISSN1551-4005
Palabras claveAdenoviridae, Deoxyribonucleases, Type II Site-Specific, DNA, DNA Damage, Fluorescent Antibody Technique, Histones, Humans, Nucleic Acid Amplification Techniques, Nucleic Acid Conformation, Saccharomyces cerevisiae Proteins
Abstract

We have developed a method to localize DNA double strand breaks (DSBs) in situ in cultured mammalian cells. Adenoviruses encoding Saccharomyces cerevisiae HO endonuclease and its cleavage site were used to induce site-specific DSBs. Rolling circle amplification (RCA), a sensitive method that allows the detection of single molecular event by rapid isothermal amplification, was used to localize the broken ends in situ. Punctate RCA signals were only seen in the cells that had been infected with both adenoviruses encoding HO endonuclease and HO cleavage site, but not in the cells mock-infected or infected with the site or endonuclease virus only. With use of a chemical crosslinker, in situ RCA and immunofluorescence (IF) can be performed simultaneously on the same sample. This methodology provides a novel approach for investigation of DNA recombination, DNA repair, and checkpoint controls in mammalian cells.

Alternate JournalCell Cycle
PubMed ID16294038
Grant ListGM31452 / GM / NIGMS NIH HHS / United States
R01CA82257 / CA / NCI NIH HHS / United States
R33CA099135 / CA / NCI NIH HHS / United States
R33CA81671 / CA / NCI NIH HHS / United States