Sample Submission GuidelinesEpiTYPER DNA methylation analysis constitutes a high-throughput DNA methylation quantification method. Making use of the MassARRAY molecular weight array gene analysis system, CD Genomics employs EpiTYPER DNA methylation technology to facilitate the precise detection and quantification of multiple CpG methylation sites. Alongside this, the accompanying EpiTYPER software provides users with convenient data analysis and reporting tools.
Introduction of EpiTYPER DNA Methylation Analysis
DNA methylation is a significant epigenetic modification, referring to the addition of methyl groups to DNA molecules, typically occurring at the 5th position of the cytosine (C) base. This modification plays a crucial role in biological processes such as genome stability, gene expression regulation, cell differentiation and tissue development.
The DNA methylation analysis technology, EpiTYPER MassARRAY, provided by Agena Bioscience (previously Sequenom Inc.), is one of the most reliable quantitative methods available today for DNA methylation analysis. EpiTYPER is a MALDI-TOF mass spectrometry-based bisulfite sequencing method that enables region-specific DNA methylation analysis in a quantitative and high-throughput fashion. The technology interrogates 10s-100s of samples and CpG sites in amplicons from 200 - 600 bp and detects down to 5% differences in methylation. It is particularly suitable for larger scale efforts to study candidate regions or to validate regions from genome-wide DNA methylation studies.
Advantages of EpiTYPER DNA Methylation Analysis
- Efficiency: Bisulfite treated DNA to data in 8 hours, Covers multiple CpGs in amplicons of up to 600 bp, Compatible with formalin-fixed paraffin embedded tissue samples
- Precise & Accurate: High precision (5% CV), High inter-laboratory reproducibility
- Sensitive: Detects down to 5% change in methylation levels
- Cost-Effective: 24-, 96-, and 384-well formats available, Multiple CpGs analyzed in one simple reaction, from amplicon as long as 200-600 bp
- Simple Workflow: No need to design CpG-specific primers, No PCR product purification required, Ideal for investigating a few or several hundred target regions, Convenient software solutions for comparison between samples
Application of EpiTYPER DNA Methylation Analysis
- Disease research
- Cancer biomarkers
- Development and differentiation studies
- Environmental exposure and epigenetics
- Drug development and personalized therapy
- Research on the association between genetic abnormalities related to inherited diseases and methylation
EpiTYPER DNA Methylation Analysis Workflow
EpiTYPER biochemistry starts with bisulfite treatment of genomic DNA, followed by PCR amplification of target regions. The reverse primers contain a T7 promoter tag. Next, in vitro RNA transcription is performed, followed by base-specific RNA cleavage. Finally, the cleavage products are analyzed using MALDI-TOF mass spectrometry (MassARRAY Analyzer). The methylated and non-methylated cytosine residues in the original genomic DNA are easily distinguished using EpiTYPER Software.
Quantitative region-specific DNA methylation analysis by the EpiTYPER™ Technology. (Kunze, 2018)
STEP 1: Assay Design
EpiDesigner is an online automated design tool for DNA methylation experiments on the MassARRAY System. Just enter your target sequences and the software determines primer designs for the most complete DNA coverage. In addition to optimized primer sequences, EpiDesigner delivers an easy-to-read graphical interpretation of the amplicons designed over your target regions, as well as annotating distinct CpG sites covered by the assays.
STEP 2: Bisulfite Treatment
Genomic DNA (gDNA) is treated with bisulfite which leads to a conversion of all unmethylated cytosines, while methylated cytosines remain unaffected.
STEP 3: PCR, In vitro transcription, and RNA Cleavage Design
The EpiTYPER Assay starts with PCR using T7-promoter-tagged reverse primers to amplify the target regions while preserving the bisulfite-induced sequence changes.
After shrimp alkaline phosphatase treatment, in vitro transcription is performed, to discard unincorporated DNA nucleotides, the T7 promoter added during PCR is used to transcribe the PCR product from the reverse strand, yielding a single-stranded RNA product. This RNA product is specifically cleaved with RNase A at uracil residues.
STEP 4: Data Acquisition and analysis
The EpiTYPER reaction products are dispensed onto a SpectroCHIP® Array (Chip). The Chip is then placed in the MALDI-TOF(Matrix Assisted Laser Desorption-Ionization Time of Flight) mass spectrometer for data acquisition, which typically requires 15-60 minutes. The results are automatically loaded into a database for data analysis with EpiTYPER software.
The matrix on the SpectroCHIP absorbs the energy of the laser and transfers it to the RNA fragments which subsequently become ionized. The ionized fragments are separated by the time it takes to arrive at the detector at the end of the mass spectrometer's flight tube under the influence of an electric field. The time of flight increases with higher mass. A fragment containing one or more CpG dinucleotides is called a CpG unit. If a CpG dinucleotide was methylated and protected from bisulfite conversion, the corresponding RNA fragment, the CpG unit, will be 16Da heavier in mass when the CpG dinucleotide was methylated, resulting in a 16Da shift in the mass spectrum. The signal detected by the mass spectrometer for either fragment is proportional to the number of fragments. The number of fragments is quantified by the surface area of the corresponding peaks in the mass spectrum. The DNA methylation percentage of a given CpG is calculated by dividing the surface area of the peak representing the methylated fragment by the total surface area of the peaks of both the methylated and unmethylated fragment.
Service Specification
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Sample requirements and preparation
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Subsequent experimental treatment
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Data Analysis
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Deliverables
- The original data
- Experimental results
- Data analysis report
- Details in Viral Genome Sequencing for your writing (customization)
CD Genomics is now providing the efficient, cost-effective, accurate, and sensitive DNA bisulfate sequencing service by using EpiTYPER MassARRAY technology for your targeted DNA methylation studies. Please contact us for more information and a detailed quote.
References:
- Claus R, Wilop S, Hielscher T, et al. A systematic comparison of quantitative high-resolution DNA methylation analysis and methylation-specific PCR. Epigenetics, 2012, 7(7): 772-780.
- Ehrich M, Nelson M R, Stanssens P, et al. Quantitative high-throughput analysis of DNA methylation patterns by base-specific cleavage and mass spectrometry. Proceedings of the National Academy of Sciences, 2005, 102(44): 15785-15790.
- Suchiman H E D, Slieker R C, Kremer D, et al. Design, measurement and processing of region-specific DNA methylation assays: the mass spectrometry-based method EpiTYPER. Frontiers in genetics, 2015, 6: 287.
- Kunze S. Quantitative region-specific DNA methylation analysis by the EpiTYPER™ Technology. DNA Methylation Protocols, 2018: 515-535.
Example of mass spectra resulting from the measurement with EpiTYPER. (Suchiman et al., 2015)
1. What happens when DNA is methylated?
When DNA undergoes methylation, it implies that the methyl group (CH3) is added to the five position of the cytosine (C) base in DNA molecules. This process is catalyzed by methyltransferase enzymes, typically appending methyl groups to CpG dinucleotides (a connection between a pair of cytosine bases and a ribonucleic acid group).
2. What is the principle of MassARRAY?
The MassARRAY Molecular Weight Array Gene Analysis System and the EpiTYPER DNA methylation analysis technology ingeniously interweaves the principles of base-specific enzymatic cleavage reactions and MALDI-TOF sequencing. The synergy of these two distinct components enhances the technical capacity, culminating in a high-accuracy, high-throughput, and high-sensitivity methodology for quantifying DNA methylation.
Just as with other sequencing methods for DNA methylation detection, the MassARRAY platform also applies the process where, upon bisulphite treatment, the unmethylated cytosines (C) within the DNA are converted to uracil (U), while methylated cytosines remain unchanged. As methylated cytosines and unmethylated cytosines (converted to U) have different molecular weights, they can be distinguished. The Utilization of 5-terminal primers with T7 promoters for PCR amplification establishes detectable products of varied molecular weights. The size and molecular weight of post-enzymatic cleavage DNA fragments is dependent on the base changes that occur after bisulphite treatment. In this way, the MassARRAY platform effectively assesses DNA methylation status.
3. What DNA methylation detection techniques are there?
DNA methylation detection encompasses a variety of methodologies including High-Performance Liquid Chromatography (HPLC), nucleic acid mass spectrometry (MassARRAY), Methylation-Sensitive Restriction Enzyme Polymerase Chain Reaction (MSRE-PCR), Bisulphite Sequencing, TaqMan Probe technique, Whole Genome Bisulfite Sequencing (WGBS), and chip-based methods, among others. Researchers must choose the most suitable technique aligning with the nature of their samples and the specificity of their research goals.
Quantification of gene-specific methylation of DNMT3B and MTHFR using sequenom EpiTYPER® Journal: Data in Brief
Impact factor: 1.263
Published: 2 December 2015
Backgrounds
DNA methyltransferase 3B (DNMT3B: NM_006892.3) and methylenetetrahydrofolate reductase (MTHFR; NM_005957.4) represent two critical genes encoding enzymes imperative to one-carbon metabolism whose disruption is progressively associated with colorectal cancer etiology. In a study involving 272 patients undergoing colonoscopy screening, DNMT3B and MTHFR - genes encoding enzymes vital to one-carbon metabolism - were quantitatively examined for DNA methylation in leukocytes through EpiTYPER®. The data derived from this study can be harnessed to provide insights for future research investigating the correlation between methylation levels at specific CpG sites and the expression of DNMT3B and MTHFR genes.
Methods
- Blood collection
- DNA extraction
- Quantification of DNA concentration
- EpiTYPER® technology
- Principal component (PC) analysis
Results
Descriptive analysis of gene-specific DNA methylation in DNMT3B and MTHFR
The methylation percentages at each informative CpG site for DNMT3B and MTHFR genes are illustrated in Figures 1 and 2, respectively. For DNMT3B, the average levels of methylation across all the informative CpG sites were 11% (SD=0.03) in events and 11% (SD=0.04) in non-events. As for MTHFR, the average methylation percentage in events was 13% (SD=0.03), whereas the non-events revealed a slightly lower average methylation percentage of 12% (SD=0.02).
Figure 1. (A) Average methylation ratio for each informative CpG site in DNMT3B according to chromosome location. CpG sites with Eigenvalues greater than 0.20 for PC1 and PC2 are indicated in red and green, respectively. (B) Diagram illustrating the transcription start site and exon 1 of the DNMT3b gene within the targeted CpG island.
Figure 2. Average percent methylation ratio for each informative CpG site in MTHFR according to chromosome location. CpG sites with Eigenvalues greater than 0.20 for PC1 and PC2 are indicated in red and green, respectively. (B) Diagram illustrating the transcription start site and exon 1 of the MTHFR gene within the targeted CpG island.
Conclusion
By employing Sequenom EpiTYPER® technology, this research quantified methylation levels at 66 and 28 CpG sites, respectively, located within the promoter regions of DNMT3B and MTHFR. There was a significant variation in methylation levels across these CpG sites. Future etiological studies exploring the role of gene-specific DNA methylation in DNMT3B and MTHFR can delve into different concepts of methylation, including principal component analysis.
Reference:
- Ho V, Ashbury J E, Taylor S, et al. Quantification of gene-specific methylation of DNMT3B and MTHFR using sequenom EpiTYPER®. Data in Brief, 2016, 6: 39-46.
