Preparation of a methylated DNA standard, and its stability on storage

Several methods are available for the determination of low levels of O-6-me thylguanine in DNA; this base arises after methylation by environmental car cinogens. The reliability of these assays is much improved by the use of a standard. We have prepared such a standard by treating calf thymus DNA with [H-3-methyl]-N-methyl-N-nitrosourea. We characterized the methylated bases by hydrolysis of a sample to nucleosides, followed by liquid chromatograph y and liquid scintillation counting of the tritium content. The level of O- 6-methylguanine was 0.6 per one million nucleotides. This base is stable, a nd its level was unchanged after storage for 5 years at -20 degrees C. The methylated DNA also contained 7-methylguanine and 3-methyladenine, which ar e slowly cleaved from the DNA on standing. The half-life for loss of 7-meth ylguanine at neutral pH was estimated to be 70 h at 39 degrees C, 460 h at 22 degrees C, 3800 h at 10 degrees C, and about 4 years at -20 degrees C.