Agena – DNA Methylation

Methylation service with Agena MassARRAY

Epigenetics describes heritable changes in gene function that occurs without a change in the DNA sequence itself. Epigenetic marks include e.g. DNA methylation and histone modifications, which are usually propagated in cell division, and may also be passed on to the next generation through germ cells. In the human genome, DNA-methylation only occurs at the consecutive base combination C phosphodiester bound G (CpG). CpGs are relatively rare in the genome and often occur in clusters located upstream of genes, so called CpG-islands. The cytosine-methyl-group bond is covalent and thus very stable.

Briefly, the EpiTYPER methodology is based on amplifying a region of interest on bisulfite-converted DNA, followed by in vitro transcription and RNase-digestion. The resulting fragments are resolved using MALDI-TOF technology on a Agena platform, where a G to A difference, resulting from differential conversion of a methylated or unmethylated CpG, can be resolved and quantified. This approach allows you to quantify methylation levels of several CpG-sites within a 200-600 bp region , thus enabling both a detailed and quantitative investigation of methylation levels.

DNA methylation quantification using Agena MassARRAY

Design of assays: Assays are designed based on sequence information provided by the customer. Primers are designated in the Epidesigner software, where a minimum of 4 analysable CpGs within the amplicon, and 4 non-CpG cytosines within the primer sequence are default parameters. The non-CpG cytosines within the primer sequence ensures exclusive amplification of bisulfite-converted DNA. The EpiTYPER procedure is directional, such that only one strand is in vitro transcribed, and in the design process both strands are investigated. The recommended amplicon size is 200-500 bp. Primer sequences are manually checked for known polymorphisms based on data from dbSNP, such that they are not variable between individuals.

Bisulfite treatment of DNA: Before PCR, the genomic DNA needs to be treated with sodium bisulfite. This conditionally converts DNA by deaminating and converting unmethylated cytosines to uracils, while leaving methylated cytosines intact. Implicitly, all cytosines that are not CpGs should be converted to uracils, while in the case of a CpG the reaction depends on its original methylation status. Samples are converted in 96-well format (ZYMO’s EZ DNA kit), using a mininum of 500 ng as input.

PCR: The PCR step both increases the template amount and, through the reverse primer, introduces a recognition site for the RNA/DNA T7 polymerase, that is used in the in vitro transcription step. Multiplexing occurs at the level of CpGs within a single amplicon and only one amplicon per well is possible.

PCR clean up: The PCR product is treated with the Shrimp alkaline phosphatase (SAP) enzyme that depohosporylates the remaining free nucleotides from the PCR.

In vitro transcription/RnaseA fragmentation: Two experimental steps are performed simultaneously; in vitro transcription and RnaseA cleavage. The T7 RNA polymerase performs directional transcription of the PCR template. The transcription mix contains rUTP, rATP, rGTP and dCTP. RNaseA recognises and cleaves at rUTP (A on the original template). In the case where a fragment contains a CpG, on the transcript level, this cytosine will correspond to an A if unmethylated and a G if methylated.

Fragment analysis: The resulting fragments are subjected to MALDI-TOF analysis on a Agena MassARRAY Analyzer, where the mass difference of a fragment between an unmethylated and methylated CpG (16 Daltons, A to G) is resolved and quantified.

Quantification of DNA methylation: The Agena’s EpiTYPER software is used to identify the mass-fragments and quantify DNA-methylation at single CpG-sites or of a CpG-unit (fragment that contains several CpGs). An in silico fragementation has been performed by the software and thus, all matched, missing, and additional peak information can be extracted.

Checking bisulfite-treated DNA quality: The quality of the genomic DNA used as input for the bisulfite treatment, is crucial for experimental success. Therefore, as a part of the validation process, bisulfite treated customer samples are tested for degradation and quality.   

Validation: Important amplicon characteristics include reproducibility, quantitativity, and succes rate. Duplicate measurements are always performed. When a customer has a region of interest, we will design several alternative assays, order primers and test for quantitativity using a methylation dilution curve. By evaluating performance, the best assay(s) will be selected for the larger project.

Control DNA and assay performance: In each analysis unit (384 well plate), we include 16 controls, including 4 fullymethylated (~100%) controls, 4 unmethylated (~0%) controls, 2 negative water controls, and 2 negative genomic DNA controls.

Important considerations:
-> This method requires a minimum amount of 500 ng per sample, which cannot be kept for longer periods of times >6 months after bisulfite treatment, due to degradation.
-> Every methylation analysis reaction is run in at least duplicate.