Detection of DNA Methylation in Genomic DNA by UHPLC-MS/MS

Methods Mol Biol. 2021;2198:79-90. doi: 10.1007/978-1-0716-0876-0_7.


DNA methylation serves to mark DNA as either a directed epigenetic signaling modification or in response to DNA lesions. Methods for detecting DNA methylation have become increasingly more specific and sensitive over time. Conventional methods for detecting DNA methylation, ranging from paper chromatography to differential restriction enzyme digestion preference to dot blots, have more recently been supplemented by ultrahigh performance liquid chromatography coupled with mass spectrometry (UHPLC-MS/MS) to accurately quantify specific DNA methylation. Methylated DNA can also be sequenced by either methylated DNA immunoprecipitation followed by sequencing (MeDIP-seq) or single-molecule real-time sequencing (SMRTseq) for identifying genomic locations of DNA methylation. Here we describe a protocol for the detection and quantification of epigenetic signaling DNA methylation modifications including, N6-methyladenine (6mA), N4-methylcytosine (4mC) and C5-methylcytosine (5mC) in genomic DNA by triple quadrupole liquid chromatography coupled with tandem mass spectrometry (QQQ-LC-MS/MS). The high sensitivity of the UHPLC-MS/MS methodology and the use of calibration standards of pure nucleosides allow for the accurate quantification of DNA methylation.

Keywords: 4mC; 5mC; 6mA; C5-methylcytosine; N4-methylcytosine; N6-methyladenine; UHPLC-MS/MS; methylated DNA.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Adenine
  • Chromatography, High Pressure Liquid*
  • DNA Methylation*
  • Epigenesis, Genetic*
  • Epigenomics* / methods
  • Epigenomics* / standards
  • Genome*
  • Reproducibility of Results
  • Tandem Mass Spectrometry*


  • Adenine