Employment of hydrolytic enzymes in the study of the level of DNA methylation

A new method for the determination of the level of DNA methylation was established. The method involves enzymatic hydrolysis of DNA by nuclease P1 and bacterial alkaline phosphatase, and separation of the resulting deoxyribonucleosides by HPLC. By this method, DNA was hydrolysed completely to the five deoxyribonucleosides and the complete base composition was determined. Pairing bases were shown to occur in similar amounts, and analysis could be performed on as little as 1 microgram of DNA with a high degree of reproducibility. Among other enzymes hitherto used in order to hydrolyze DNA, micrococcal nuclease, phosphodiesterase II and nuclease P1 have been shown to cause deamination of deoxyadenosine, while deoxyribonuclease I, phosphodiesterase I and bacterial alkaline phosphatase have been shown to be sensitive to contamination by RNA, and to release 5-methyldeoxycytidine at a slower rate than the other four deoxyribonucleosides. Neither of these effects was seen with the new method.