Determination of N6-Methyladenine in DNA of Mammals and Plants by Dpn I Digestion Combined with Size-Exclusion Ultrafiltration and Mass Spectrometry Analysis

Abstract

AbstractDNA adenine methylation (N6-methyladenine, 6mA) widely exists in bacteria and regulates various biological processes including gene transcription and restriction-modification system. In recent years, it has been reported that 6mA is also present in genomic DNA of higher eukaryotes. However, the accurate determination of 6mA in DNA of higher eukaryotes is challenging owing to the potential contamination of bacterial DNA that normally contains abundant 6mA. Therefore, unambiguous detection and quantification method is required for the accurate analysis of 6mA in DNA of higher eukaryotes. We developed a method by Dpn I digestion combined with size-exclusion ultrafiltration and mass spectrometry analysis to determinate 6mA in DNA of higher eukaryotes, including mammals and plants. The contamination of potential bacterial DNA can be efficiently removed from the extracted eukaryotic DNA, which enables the accurate and confident determination of 6mA in DNA of higher eukaryotes.KeywordsDNA adenine methylationN6-methyladenineDpn I digestionSize-exclusion ultrafiltrationLiquid chromatography-mass spectrometry