Massarray agena12/27/2023 ![]() ![]() Duplicate measurements are always performed. Validation: Important amplicon characteristics include reproducibility, quantitativity, and succes rate. Therefore, as a part of the validation process, bisulfite treated customer samples are tested for degradation and quality. An in silico fragementation has been performed by the software and thus, all matched, missing, and additional peak information can be extracted.Ĭhecking bisulfite-treated DNA quality: The quality of the genomic DNA used as input for the bisulfite treatment, is crucial for experimental success. 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). 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.įragment 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. RNaseA recognises and cleaves at rUTP (A on the original template). The transcription mix contains rUTP, rATP, rGTP and dCTP. The T7 RNA polymerase performs directional transcription of the PCR template. In vitro transcription/RnaseA fragmentation: Two experimental steps are performed simultaneously in vitro transcription and RnaseA cleavage. PCR clean up: The PCR product is treated with the Shrimp alkaline phosphatase (SAP) enzyme that depohosporylates the remaining free nucleotides from the PCR. Multiplexing occurs at the level of CpGs within a single amplicon and only one amplicon per well is possible. 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. Samples are converted in 96-well format (ZYMO’s EZ DNA kit), using a mininum of 500 ng as input. 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. This conditionally converts DNA by deaminating and converting unmethylated cytosines to uracils, while leaving methylated cytosines intact. Primer sequences are manually checked for known polymorphisms based on data from dbSNP, such that they are not variable between individuals.īisulfite treatment of DNA: Before PCR, the genomic DNA needs to be treated with sodium bisulfite. The recommended amplicon size is 200-500 bp. The EpiTYPER procedure is directional, such that only one strand is in vitro transcribed, and in the design process both strands are investigated. The non-CpG cytosines within the primer sequence ensures exclusive amplification of bisulfite-converted DNA. 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. DNA methylation quantification using Agena MassARRAYĭesign of assays: Assays are designed based on sequence information provided by the customer. 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. 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. The cytosine-methyl-group bond is covalent and thus very stable.īriefly, the EpiTYPER methodology is based on amplifying a region of interest on bisulfite-converted DNA, followed by in vitro transcription and RNase-digestion. CpGs are relatively rare in the genome and often occur in clusters located upstream of genes, so called CpG-islands. In the human genome, DNA-methylation only occurs at the consecutive base combination C phosphodiester bound G (CpG). 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. Familial Hypercholesterolemia (FH) analysisĮpigenetics describes heritable changes in gene function that occurs without a change in the DNA sequence itself.Increased Efficiency with New Assay for Zygosity Analysis.Development of a fast and cost-effective genetic diagnostic method for familial hypercholesterolemia in Sweden. ![]()
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