Since its discovery in 1950, researchers have been investigating 5-methylcytosine (5-mC), the highly-present epigenetic modification known as DNA methylation. Measuring the total amount of 5-mC or 5-hmC (5-hydroxymethylcytosine) provides insight to researchers to gain understanding into the gene expressions that promote or inhibit significant biological processes. These findings have been found to allow researchers to find biomarkers for diseases and other epigenetic changes that are affected by gene expression.
Throughout the time of these discoveries, a variety of global DNA methylation assays have been developed and utilized as new technology and processes are created to measure the quantitation of overall 5-mC content in DNA samples more accurately and faster than past methods. Today, a wide range of different assays exist, each with their own upsides, limitations, throughput capabilities and input recommendations.
Here we will look at the differences between some of the most widely available methods of global DNA methylation including the advantages, disadvantages and capabilities for the following methods: ELISA, LINE-1, HPLC, LC-MS/MS, M.Sssl Acceptance Assay, LUMA, Bisulfite Sequencing of Repetitive Elements, and End-specific PCR.
Global DNA Methylation Method Comparison
|DNA is denatured and then immobilized and incubated with 5-mC monoclonal antibody. Quantitation is based on microplate spectrophotometer staining intensity reading after incubation with fluorescein-conjugated secondary antibody.
||• Commercial kits available
• Simple protocol
• Can map DNA methylation location on chromosomes in individual cells (e.g. juxtacentromeric regions)
||Between 20-1000 ng for different commercial kits
||• Reliant on quality of 5-mC monoclonal antibody, fluorescein-conjugated secondary antibody, and successful washing
Nuclear Element 1 (LINE-1) Methylation Analysis
|Analyzes methylation status of LINE-1 retrotansposons and this data is extrapolated upon for global DNA methylation status.
||• Uses methylation of LINE-1 repeats (which comprise 17-20% of the mammalian genome) to quantify global DNA methylation|
||• Assay procedure takes up to 5 hours to complete
• DNA hybridization step required during process increases chances of error
Performance Liquid Chromatography (HPLC)
|Total genomic DNA is degraded to individual deoxyribonucleotides and RNA is removed via ribonuclease treatment. Then converted to deoxyribonucleosides via alkaline phosphatase treatment. Reverse phase HPLC separates products and then external standards or UV absorbance are used for quantification.
||• Highly quantitative
• Preferred quantitative technique prior to LC-MS/MS assays
||• Internal standards are difficult to incorporate
• Co-elution of contaminants with DNA may reduce accuracy
Chromatography-tandem Mass Spectrometry (LC-MS/MS)
|Internal standards are spiked, but the procedure is the same as
HPLC. Quantitation using mass spectrometry.
• Absolute quantification
||• Costly to set up instruments
• Costly to make or buy internal standards
• Expertise in complex mass spec procedure necessary
|Genomic DNA is incubated with DNA methyltransferase (M.Sssl) and methyl donor S-adenosylmethionine (SAM). DNA is then immobilized on nitrocellulose paper and scintillation counter then measures ionizing radiation.
||• Uncomplicated and quick protocol
• Reliant on quality of DNA methyltransferase M.SssI and stability of S-adenosylmethionine (SAM)
• Intrassay and interassay variability may be large
• Radiation required
• Difficult to compare results across labs
|Luminometric Methylation Assay (LUMA)
||Methylation-sensitive and insensitive restriction enzymes (HpaII, MspI, and EcoRI) are used to cleave DNA followed by polymerase extension assay by pyrosequencing.
• Strong internal control using EcoRI
||• Sequence specific; may not entirely represent global DNA methylation
• Difficult to compare results against other methods
|Bisulfite Sequencing of Repetitive Elements
||DNA is bisulfite-converted and repetitive elements are amplified by real-time PCR (RT-PCR) or standard PCR. Quantification by pyrosequencing or combined bisulfite restriction analysis (COBRA).
||• Relatively quantitative for each repeat element
||< 100 ng
||• Inconclusive about whether methylation levels at repeat elements indicate global DNA methylation
• High inter-assay variability
• Bisulfite treatment may cause errors due to the frequent conversion of methylated cytosines into thymine at repetitive elements
|Methylation-sensitive restriction enzyme digestion followed by RT-PCR and fluorescence-based readout are used to quantitatively measure level of unmethylated LINE-1, Alu and LTR.
• Evidence indicates it may be a poor surrogate measure of global DNA methylation
• Restricted to human DNA samples only
• High background noise from highly heterogeneous regions
• Can only assess certain subsets of LINE and Alu repeats