An Improved Sample Preparation Method for the Quantitative HPLC Determination of 5-Methyl-Deoxycytidine in Animal Tissue DNA

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Abstract
Techniques are presented for the purification of DNA from mammalian tissues, its enzymatic hydrolysis to deoxyribonucleosides and the separation and quantification of these by high pressure liquid chromatography (HPLC). The method is used to quantify 5-niethyldeoxycytidine (5MdC) and deoxycytidine (dC) in DNA. From this data the molar %5MdC, i.e. 100 × 5MdC/(dC + 5MdC), is calculated for DNA. The precision of the method matches or exceeds that of other published HPLC methods for quantifying the %5MdC. The DNA obtained is extremely clean, and the enzymatic hydrolysis provides deoxyribonucleosides without contamination so there are no extraneous peaks in the region of interest, and peaks that are 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, drugs or carcinogens.