Estimation of DNA damage and repair in tissues of gamma-irradiated animalsusing the polymerase chain reaction

Damage and repair of DNA isolated from brain and spleen of gamma-irradiated rats were assayed using the polymerase chain reaction (PCR) method. Damage produced by gamma-radiation in DNA in cells of these tissues of exposed an imals was shown to block PCR with the Tth polymerase, This blockage was not ed as a decrease in the level of amplification of the fragments of a transc ribed gene (beta-actin), an inducible gene (p53), and a nontranscribed one (IgE, heavy chain). The most pronounced decrease in the amplification of th e gene fragments was observed on the DNA template isolated from rats immedi ately after their gamma-irradiation. When DNA was isolated 0.5-5.0 h after exposure, the amplification level was restored, no matter what transcriptio n activity the genes possessed. For comparison, we used in PCR in vitro gam ma-irradiated DNA as well as DNA templates with UV-damage, 8-oxy-2'-deoxygu anosine (8-O-dG), and apurinic sites (AP-sites). We found that gamma- and U V-irradiated DNA as well as DNA with AP-sites blocked the Tth polymerase in PCR, whereas 8-O-dG did not effect the level of PCR amplification of gene fragments. The observed changes in the level of PCR amplification of genes on the DNA template from tissues of gamma-irradiated animals are due to var ious radiation-induced lesions capable of blocking the Tth polymerase. The results show that the PCR method call be used for assaying the integral DNA damage and repair in cells from irradiated animals.