Thymine Dimer Products

Non-radioactive labeling of DNA is typically based on the enzymatic incorporation of modified nucleotides, carrying a small chemical moiety such as biotin, digoxigenin or fluorescein. These tags are subsequently detected by specific reagents such as streptavidin or a specific antibody coupled to a signal-producing enzyme. Although very efficient and reliable, labeling by in vitro polymerization is time-consuming, expensive, and may require various post-label purification steps to remove an excess of unincorporated precursors. An alternative strategy for DNA labeling, is based on the UV-induced formation of cyclobutane thymine dimers. Several methods have been described for the detection of thymine dimers, which are based on chromato-graphic analysis, and on biochemical analysis with endonucleases specific for UV-irradiated DNA. In addition, methods utilizing antibodies specific for pyrimidine dimers and other UV-induced DNA lesions have evolved, which permit the study of the induction and repair of these lesions without the requirement of in vivo radiolabeling of DNA. Photoimmunodetection, is a rapid, reliable and low-cost supplement to existing methods for nonradioactive DNA labeling. It enables a sensitive and non-radioactive method for labeling, detection, and quantification of high molecular weight (HMW) DNA fragments. The method is based on the introduction of thymine dimers into DNA after separa-tion by pulse field gel electrophoresis (PFGE), followed by detection with thymine dimer specific antibodies. The method does not require any enzymatic or chemical manipulation of the DNA sample. Monoclonal anti-bodies reacting specifically with thymine dimer, facilitate investigations on the apoptotic process and the role of UV-induced pyrimidine dimers in the process of photocarcinogenesis.