Article written by: Sylwia Adamczyk, Joanna Chojak-Koźniewska, Anna Poimala, Sannakajsa Velmala, Bartosz Adamczyk
Abstract: DNA (Deoxyribonucleic acid) methylation is one of the epigenetic modifications of DNA, acting as a bridge between genotype and phenotype. Thus, disruption of DNA methylation pattern has tremendous consequences for organism development. Current methods to determine DNA methylation suffer from methodological drawbacks like high requirement of DNA and poor reproducibility of chromatograms. Here we provide a fast and reliable method using high-pressure liquid chromatography (HPLC)-ultraviolet (UV) detector and even more sensitive one with HPLC- mass spectrometry (MS) and we test this method with various plant and fungal DNA isolates. We optimized the preparation of the DNA degradation step to decrease background noise, we improved separation conditions to provide reliable and reproducible chromatograms and conditions to measure nucleotides in HPLC-MS. We showed that global DNA methylation level can be accurately and reproducibly measured with as little as 0.2 µM for HPLC-UV and 0.02 µM for HPLC-MS of methylated cytosine.