Determination of DNA methylation by COBRA: A comparative study of CGE with LIF detection and conventional gel electrophoresis

Abstract

DNA methylation as an epigenetic modification of the human genome is under emphatic investigation. Several studies have demonstrated a role of DNA methylation in oncogenesis. In conjunction with histone modifications, DNA methylation may cause the formation of heterochromatin and thus mediate the inactivation of gene transcription. It is important to develop methods that allow for an accurate quantification of the amount of DNA methylation in particular DNA regions, to gain information concerning the threshold of methylation levels necessary for gene inactivation. In this article, a CGE method with on-column LIF detection using SYBR Green is compared with a conventional slab-gel electrophoresis. We thus investigate the validity to analyze DNA methylation in the samples of a combined bisulfite restriction analysis. It is demonstrated that CGE is superior to gel electrophoresis in means of linearity, precision, accuracy, automatization (high throughput), and sample consumption. However, gel electrophoresis is easier to perform (simple devices, no PC usage), and the running costs are comparatively low. A further advantage of CGE is the sparse use of toxic compounds (MeOH and SYBR Green), whereas gel electrophoresis is performed in polyacrylamide gels with ethidium bromide staining.

Adaption of COBRA Method for The Investigation of Nutrition-Based DNA Methylation Mechanism in Escherichia coli

“…As a result, the data obtained from our present study indicate that COBRA technique can be used effectively in prokaryotic systems in the investigation of DNA methylation profile changes due to epigenetic events, similar to the applications of many eukaryotic systems in the literature (Xiong and Laird, 1997;Goedecke et al, 2009). In this respect, our present study differs from other applications performed with eukaryotic systems and shows originality.…”

Vitamin and antioxidant rich diet increases MLH1 promoter DNA methylation in DMT2 subjects

“…The Bst UI restriction site (CGCG) in the MLH1 promoter analyzed by combined bisulfite restriction analysis (COBRA) was hypomethylated at all time points and possible minor methylation shifts were not detectable (see Additional file 1) [22]. DNA methylation differences between baseline (T0) and after 8 weeks of the intervention (T1) were not significantly different in the intervention groups.…”

Phosphorus and osmium as elemental tags for the determination of global DNA methylation—A novel application of high performance liquid chromatography inductively coupled plasma mass spectrometry in epigenetic studies

“…This parameter is defined as the ratio between methylated cytosines and total amount of cytosine bases in DNA. Several analytical procedures have been reported, based on different DNA digestion protocols followed by chromatographic or electrophoretic separation [11,12,14,[18][19][20][21][22][23]. The detection tools employed in those studies include UV/vis [12,14,22], molecular mass spectrometries [11,20,21] and measurements of laser-induced fluorescence [23].…”