Global DNA Methylation: Overview and Assays
Below is a collection of useful links, materials, and descriptions related to epigenetics, DNA methylation, and global DNA methylation analysis. These resources for global DNA methylation can help researchers who are investigating epigenetics in general as well as those specifically measuring genome-wide 5-mC (5-methylcytosine) or 5-hydroxymethylcytosine (5-hmC) levels. They offer an overview of DNA methylation, the fundamentals of epigenetics and how it relates to the development of various diseases, and an introduction to a highly accurate and cost-effective method used to measure global DNA methylation.
Global 5-mC DNA Methylation Quantification Methods
High Performance Liquid Chromatography (HPLC)
Prior to LC-MS/MS assays, the HPLC technique was the preferred method for quantifying global DNA methylation. HPLC provides an absolute measurement of 5-mC in samples of DNA by enzymatic hydrolysis of genomic DNA. This degrades the input DNA to individual deoxyribonucleotides. RNA is then removed to avoid contamination via treatments with ribonuclease and the deoxyribonucleotides are then converted to deoxyribonucleosides. Isocratic reversed-phase HPLC is used to separate the products and external standards or UV absorbance will quantify 5-mC. A significant downfall to this method, however, is that it typically requires large amounts of sample DNA, which may often be hard to come by, and optimizing the assay’s protocol requires experimental expertise.
Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS)
Liquid chromatography tandem mass spec (LC-MS/MS) is considered the “gold standard” for measuring global DNA methylation, but it is a complex method and requires access to costly equipment. The procedure is similar to HPLC, as it digests and separates deoxyribonucleotides, but a highly specialized skill set is required for analysis by mass spectrometry. Quantification is incredibly sensitive and several variations that have been designed which vary in the type of standards used for quantification, the solvents, and the HPLC columns.
LUminometric Methylation Assay (LUMA)
LUMA relies on the combination of cleaving the DNA via methylation-sensitive restriction enzymes and pyrosequencing. It is quantitative and thought to be highly reproducible. However, it is sequence specific, and the scientific literature does not conclusively point to whether this method entirely represents global DNA methylation.
M.SssI Acceptance Assay
M.SssI acceptance assay is a simple and fast procedure that uses CpG DNA methyltransferase M.SssI and methyl donor S-adenosylmethionine that is incubated with genomic DNA. Radioactivity is needed for analysis and evidence shows that large intra- and interassay variability can occur if the enzyme activity is not successful and if stability of the methyl donor SAM is sub-optimal.
Global DNA Methylation 5mC Quantification ELISA
This video explains the use of an ELISA-like method to measure levels of global DNA methylation in DNA samples obtained from a broad range of species. It is a cheaper, more convenient alternative than mass-spectrometry or chromatography-based techniques. Watch Video
|Overcoming Limitations of the Important Global DNA Methylation Assay
Accurate measurement of total 5-methylcytosine (5-mC) can be difficult with numerous global DNA methylation assays available, each with its own advantages and disadvantages. Take a look at Overcoming Limitations of the Important Global DNA Methylation Assay, a white paper written by epigenetic scientists that describes new and old methods, how to overcome assay limitations, and the optimal assay for investigating global DNA methylation levels.
Overview of Epigenetics and DNA Methylation
Perfect for an overview of epigenetics that touches on the evolving landscape of epigenetic research over time, lifestyle choices that can impact epigenetic makeup, and clinical applications such as cancer, immunity-related disorders, and neurodegenerative diseases.
Getting Started in Epigenetic Research
Introduction to trends in epigenetic research describes the easiest ways to begin research in the field of epigenetics. Provides resources and tools needed for global DNA methylation analysis and more.
Gene Expression and Cancer Epigenetics
Interactive introductory tool for understanding and visualizing the connection between genes, mRNA, and the production of proteins. Engaging content discussing the way the environment impacts a cell’s epigenome, DNA methylation, gene expression, and cancer. Use the tool to control epigenetic tags see the resulting change in the shape of the gene.
DNA Methylation Overview
A description of the epigenetic mechanism DNA methylation/DNA demethylation, the various methods used for studying these marks such as Enzyme Linked Immunosorbent Assay (ELISA), Bisulfite Conversion, and Methylated DNA Immunoprecipitation, and a brief introduction to 5-hydroxymethylcytosine (5-hmC) and DNA hydroxymethylation.
A comprehensive description of DNA methylation, including the implications of global DNA methylation on cancer, aging, atherosclerosis, and exercise. Also lists assays currently used in scientific research for detecting DNA methylation such as Methylation-Specific PCR (MSP), Mass Spectrometry, whole genome bisulfite sequencing, methylated DNA immunoprecipitation (MeDIP), and more.