Analysis of modified bases in DNA by stable isotope dilution gas chromatography-mass spectrometry: 5-methylcytosine.
A sensitive assay for 5-methylcytosine in DNA has been developed based on stable isotope dilution gas chromatography-mass spectrometry with selected ion monitoring. 5-[( 2H3]-Methyl)cytosine and [methyl-2H3]thymine have been synthesized as internal standards for analysis of DNA following acid digestion, conversion of pyrimidines to volatile t-butyldimethylsilyl derivatives, and separation in 3 min by gas chromatography. Submicrogram amounts of DNA have been analyzed for 5-methylcytosine content in the range 0.02-1.5 mol%. The estimated limit of quantitative measurement is 0.3 pmol of methylated base in a DNA hydrolysate. The method is compared with other techniques for quantitative measurement of methylated bases in DNA, and 5-methylcytosine levels and precision of analysis for calf thymus, pBR322, and phi X-174 DNAs are reported and compared with literature values. The method can readily be adapted to the accurate high-sensitivity analysis of other methylated bases in DNA.
D044503:5-Methylcytosine; D000818:Animals; D003596:Cytosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000592:standards; D004247:DNA / Q000032:analysis*; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D012015:Reference Standards; D013950:Thymus Gland / Q000378:metabolism
D044503:5-Methylcytosine; D003596:Cytosine; D004247:DNA