47] Analysis of 5-methylcytosine in DNA by isotope dilution gas chromatography—mass spectrometry

Abstract

Publisher Summary This chapter discusses the analysis of 5-methylcytosine in DNA by isotope dilution gas chromatography-mass spectrometry. 5-Methylcytosine is a minor base present in DNA from many organisms, and in higher eukaryotes, there is, in many cases, a general correspondence between hypomethylation and gene activation. For cases where an accurate and unbiased direct chemical measurement of the amount of 5-methylcytosine in DNA is required, a sensitive assay has been developed based on stable isotope dilution gas chromatography-mass spectrometry (GC/MS) with selected-ion monitoring. The basic structure of the assay consists of the addition of isotopically labeled thymine and 5-methylcytosine to the Deoxyribo-nucleic acid (DNA) prior to hydrolysis, to serve as internal standards for measurement of the corresponding DNA bases as their tert-butyldimethylsilyl (TBDMS) derivatives, and then analysis by capillary column GC/MS with selected-ion monitoring. Microscale hydrolysis and derivatization are performed in the same tube, and the bases are analyzed directly from the reaction solution. No clean-up is required past the hydrolysis step thus minimizing the potential for sample loss.