Use of high-performance liquid chromatography to quantitate thymine-containing pyrimidine dimers in DNA

We have developed two high-performance liquid chromatographic systems for the measurement of pyrimidine dimers in hydrolysates of DNA. Normal-phase chromatography on an NH2 column in methanol-ethyl acetate (3.97) at an elution rate of 2.0 ml/min allowed quantitation of thymine-containing (thymine-thymine plus thymine-uracil) pyrimidine dimers at levels as low as 0.1% of the total radioactivity as thymine in DNA. This system was unaffected by the presence of up to 1 mg of contaminating protein (bovine serum albumin) or 40 micrograms of DNA in hydrolysates prepared for chromatography. Reversed-phase chromatography on a muBondapak C18 column allowed measurement of thymine-thymine dimers at concentrations as low as 0.02% of the total radioactivity. With 0.1% tetrahydrofuran in water as the solvent at a flow-rate of up to 0.6 ml/min, thymine-thymine, thymine-uracil, and uracil-uracil dimers were completely resolved. We were not able to quantitate the latter two dimeric forms, however, owing to the presence of other radioactive components of undefined origin that eluted concomitantly with the uracil-containing dimers.