Evaluation of UV-C induced changes in Escherichia coli DNA using repetitive extragenic palindromic-polymerase chain reaction (REP-PCR).
Ultraviolet radiation is an efficient inactivation method for a broad range of bacteria, viruses and parasites. Inactivation of microorganisms by UV-B and UV-C radiation is driven through modifications in their genomic DNA, being the most stable DNA-lesions different kinds of pyrimidine dimers (PDs) (e.g., cyclobutane pyrimidine dimers (CPDs) and other photoproducts). Taking into account that these modifications inhibit the DNA polymerization in vivo as well as in vitro, in the present work the usefulness of the REP-PCR assay to detect UV-induced changes in the Escherichia coli DNA was evaluated. In vitro amplification of DNA extracted at different times after UV treatment showed a disappearance of amplicons of higher size as time of treatment increases. When the bacteria were let to progress through their dark repair process, modifications in the electrophoretic patterns by REP-PCR were observed again. Amplified bacterial DNA tended to recover the profile showed at the beginning of treatment. In addition, the reappearance of bands of higher molecular size was associated to an increase in their signal intensity probably due to a higher amplification rate. Results of REP-PCR were correlated to the colony-forming ability of E. coli. It was concluded that REP-PCR appears as a rapid, robust, useful complementary methodology to monitor the impact of UV irradiation--at a molecular level--on the inactivation and the mechanisms of repair, applicable on a broad spectrum of microorganisms.
Trombert
Alejandro
A
Instituto de Desarrollo Tecnológico para la Industria Química, INTEC (Universidad Nacional del Litoral and CONICET), Güemes 3450, Santa Fe, Argentina.
Irazoqui
Horacio
H
Martín
Carlos
C
Zalazar
Fabián
F
D015169:Colony Count, Microbial; D004260:DNA Repair; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism / Q000528:radiation effects*; D003624:Darkness; D003720:Densitometry; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics* / Q000528:radiation effects; D016680:Genome, Bacterial / Q000235:genetics; D050296:Microbial Viability / Q000528:radiation effects*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D014466:Ultraviolet Rays
D004269:DNA, Bacterial