Validation of phage T7 biological dosimeter by quantitative polymerase chain reaction using short and long segments of phage T7 DNA.
Phage T7 can be used as a biological dosimeter; its reading, the biologically effective dose (BED), is proportional to the inactivation rate |ln (n/n0)|. For the measurement of DNA damage in phage T7 dosimeter, a quantitative polymerase chain reaction (QPCR) methodology has been developed using 555 and 3826 bp fragments of phage T7 DNA. Both optimized reactions are so robust that an equally good amplification was obtained when intact phage T7 was used in the reaction mixture. In the biologically relevant dose range a good correlation was obtained between the BED of the phage T7 dosimeter and the amount of ultraviolet (UV) photoproducts determined by QPCR with both fragments under the effect of five various UV sources. A significant decrease in the yield of photoproducts was detected by QPCR in isolated T7 DNA and in heated phage compared with intraphage DNA with all irradiation sources. Because the yield of photoproducts was the same in B, C and A conformational states of T7 DNA, a possible explanation for modulation of photoproduct frequency in intraphage T7 DNA is that the presence of bound phage proteins induces an alteration in DNA structure that can result in increased induction of photoproducts.
Hegedüs
M
M
Institute of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary.
Módos
K
K
Rontó
Gy
G
Fekete
A
A
D017123:Bacteriophage T7 / Q000235:genetics*; D001483:Base Sequence; D017931:DNA Primers; D004279:DNA, Viral / Q000235:genetics* / Q000528:radiation effects; D016133:Polymerase Chain Reaction / Q000379:methods*; D014466:Ultraviolet Rays
D017931:DNA Primers; D004279:DNA, Viral