Measurement of incorporation of bromodeoxyuridine into DNA by high performance liquid chromatography using a novel fluorescent labelling technique.

Abstract

5'-Bromo-2-deoxyuridine (BUdR) is a halogenated pyrimidine analogue that is an efficient radiosensitizer through its incorporation into DNA in place of thymidine. Radiosensitization is proportional to percentage replacement and we present here a novel derivatization technique that specifically labels the thymidine and BUdR with 4-bromomethyl-7-methoxycoumarin (BrMMC) to give the highly fluorescent coumarin derivatives which are quantitated using high performance liquid chromatography (HPLC). This allows for a simple single-stage DNA hydrolysis and sensitive peak detection. Data are presented showing the incorporation with time of BUdR into the DNA of Chinese hamster V79 cells. Attention is also drawn to the care needed in the selection of enzymes required for DNA digestion.