Microwave assisted DNA hydrolysis for global methylation analysis by gas chromatography/tandem mass spectrometry

Abstract

The analysis of the global DNA methylation, calculated as the percentage of 5-methylcytosine (5mC) over the total sum of cytosines, is a well stablished biomarker for monitoring large scale epigenetic events in organisms. DNA purification, hydrolysis, separation and detection methods are critical steps to determine this biomarker. In the present work is proposed a robust procedure for DNA acid-hydrolysis assisted by microwave that provides identical DNA methylation patterns that enzymatic hydrolysis and better release of 5mC than acid classic method. The quantification was performed using a gas chromatographer coupled to a mass spectrometer with triple quadrupole as mass analyzer (GC-TQ-MS/MS) using multiple reaction monitoring (MRM) mode for the trimethylsilyl-derivates of nucleobases; following the transitions of 254→238, 240→170 and 254→238, 254→184 (m/z) for C and 5mC respectively, was achieved a limit of detection of 0.46 fmol for C and 0.41 fmol for 5mC. The proposed procedure is capable of determine 0.004% of 5mC in 50 ng of DNA in a chromatographic time of 10 minutes, being a good alternative to LC-MS/MS analysis.