AN IMPROVED SAMPLE PREPARATION METHOD FOR THE QUANTITATIVE HPLC DETERMINATION OF 5-METHYLDEOXYCYTIDINE IN ANIMAL TISSUE DNA

Techniques are presented for the purification of DNA from mammalian ti ssues, its enzymatic hydrolysis to deoxyribonucleosides and the separa tion and quantification of these by high pressure liquid chromatograph y (HPLC). The method is used to quantify 5-methyldeoxycytidine (5MdC) and deoxycytidine (dC) in DNA. From this data the molar %5MdC, i.e. 10 0 x 5MdC/(dC + 5MdC), is calculated for DNA. The precision of the meth od matches or exceeds that of other published HPLC methods for quantif ying the %5MdC. The DNA obtained is extremely clean, and the enzymatic hydrolysis provides deoxyribonucleosides without contamination so the re are no extraneous peaks in the region of interest, and peaks that a re obtained have sufficient area to eliminate errors from variation in integration. The %5MdC is a quantitative and absolute measure of the genome wide DNA methylation. This method is suitable to quantify small changes in mammalian enzymatic DNA methylation due to age, diet, drug s or carcinogens.