In situ detection of specific DNA double strand breaks using rolling circle amplification.
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.
Li
Jia
J
Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
Young
C S H
CS
Lizardi
Paul M
PM
Stern
David F
DF
D000256:Adenoviridae / Q000472:pathogenicity; D004247:DNA / Q000032:analysis / Q000737:chemistry* / Q000235:genetics*; D004249:DNA Damage / Q000235:genetics*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006657:Histones / Q000378:metabolism; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009690:Nucleic Acid Conformation; D029701:Saccharomyces cerevisiae Proteins
C468783:H2AX protein, human; D006657:Histones; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; C530845:HO protein, S cerevisiae; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific