5-Bromouracil (BrU) is photoreactive toward near UVB photons and can be introduced into genomic DNA during its biosynthesis in cells. However, PCR seems to be a simpler approach, which can be used to obtain labeled DNA similar to that synthesized within the cell. In the current work, PCR has been employed and optimized in order to substitute all thymines (besides those present in starters) with BrU in the dsDNA fragment of 80 base pairs (bp) in length. The modified oligonucleotide was irradiated with 300 nm photons in a buffered aqueous solution (pH = 7) and digested with a cocktail of enzymes specific to the phosphodiester bond cleavage. Initially, the extent of damage in the intact photolyte was measured with DHPLC. Then, the digested reaction mixture was subjected to HPLC and MS analyses and, in addition to the formation of 5-bromo-2'-deoxuyridine, which proves the occurrence of single strand breaks (SSBs) due to irradiation, UAU and UAC dimers were found, whose molecular structure was confirmed by MS/MS analysis. Although the abundance of such tandem lesions is lower than that of the SSB type, they pose a potent threat to genome integrity. Thus, our findings shed new light on the photosensitizing properties of BrU toward DNA.