Artificial plasmid labeled with 5-bromo-2'-deoxyuridine: a universal molecular system for strand break detection.
DNA strand breaks (SBs) are among the most cytotoxic forms of DNA damage, and their residual levels correlate directly with cell death. Hence, the type and amount of SBs is directly related to the efficacy of a given anticancer therapy. In this study, we describe a molecular tool that can differentiate between single (SSBs) and double (DSBs) strand breaks and also assess them quantitatively. Our method involves PCR amplification of a linear DNA fragment labeled with a sensitizing nucleotide, circularization of that fragment, and enzymatic introduction of supercoils to transform the circular relaxed form of the synthesized plasmid into a supercoiled one. After exposure of the molecule to a damaging factor, SSB and DSB levels can be easily assayed with gel electrophoresis. We applied this method to prepare an artificial plasmid labeled with 5-bromo-2'-deoxyuridine and to assay SBs photoinduced in the synthesized plasmid.
Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308 Gdansk (Poland).
Agnieszka
A
Zylicz-Stachula
Katarzyna
K
Polska
Piotr
P
Skowron
Janusz
J
Rak
D001973:Bromodeoxyuridine / Q000032:analysis*; D004247:DNA / Q000032:analysis / Q000235:genetics*; D053960:DNA Breaks / Q000528:radiation effects; D010957:Plasmids / Q000032:analysis* / Q000235:genetics; D016133:Polymerase Chain Reaction; D011838:Radiation-Sensitizing Agents / Q000032:analysis*
D011838:Radiation-Sensitizing Agents; D004247:DNA; D001973:Bromodeoxyuridine
DNA circularization; DNA damage; DNA strand breaks; UV irradiation; sensitizers