Preparation of a methylated DNA standard, and its stability on storage.
Several methods are available for the determination of low levels of O(6)-methylguanine in DNA; this base arises after methylation by environmental carcinogens. 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 [(3)H-methyl]-N-methyl-N-nitrosourea. We characterized the methylated bases by hydrolysis of a sample to nucleosides, followed by liquid chromatography 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, and its level was unchanged after storage for 5 years at -20 degrees C. The methylated DNA also contained 7-methylguanine and 3-methyladenine, which are slowly cleaved from the DNA on standing. The half-life for loss of 7-methylguanine 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.
Osborne
M R
MR
Molecular Carcinogenesis Section, Institute of Cancer Research, Haddow Laboratories, Cotswold Road, Sutton, Surrey SM2 5NG, UK. martino@icr.ac.uk
Phillips
D H
DH
D004355:Drug Stability; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000592:standards
C044502:3-methylguanine; D006147:Guanine; C008450:7-methylguanine; C008449:O-(6)-methylguanine