PyroMark CpG Assays are the only genomewide, predesigned methylation assays for Pyrosequencing analysis that use a tailored design algorithm for highly specific assay design and successful CpG methylation results. Our upgraded assay database now includes over 84,000 individual assays for the human, mouse, and rat genomes. We also now offer predesigned assays for validating results from methylation arrays such as the HumanMethylation450K BeadChip array.
Features and benefits
PyroMark CpG Assays are predesigned DNA methylation assays for Pyrosequencing analysis, delivering highly specific quantification of CpG methylation for individual or multiple CpG sites. Each ready-to-use assay includes PCR and sequencing primers that have been designed by a rigorously tailored algorithm, along with an assay setup file and information about the CpG sites covered by the assay.
Benefits of PyroMark CpG Assays include:
- Advanced assay design algorithms provide optimized results
- Comprehensive coverage of CpG sites in the human, mouse, and rat genomes
- Convenient download of run files and analysis settings
- Easy-to-order predesigned assays for gene-specific or methylation array CpG sites
- Top performance from a cost-effective solution
Highly successful PCR and Pyrosequencing
Successful methylation quantification by Pyrosequencing requires highly specific and unbiased PCR amplification. Bisulfite converted DNA is a less complex template and can be challenging to amplify specifically and at high yields. All primers in PyroMark CpG Assays have been checked against the entire human, mouse, or rat bisulfitome and are delivered at optimal concentrations for high performance. Together with the unique DNA protect technology of EpiTect Fast Bisulfite Conversion Kits and the carefully formulated reagents of the PyroMark PCR Kit, these predesigned assays ensure high yield and quality of sequencing template and increase reliability of downstream Pyrosequencing analyses.
Evaluation of PCR performance for 310 randomly selected PyroMark CpG Assays showed that 305 assays (98%) produced the predicted PCR amplicon, suitable for use as Pyrosequencing template. From these assays, 281 (92%) provided successful Pyrosequencing results (see figures High PCR success rate with bisulfite converted DNA
and High Pyrosequencing success rate of predesigned PyroMark CpG Assays
). These high-performing assays eliminate the need for tedious assay optimization and shorten the time to reliable results.
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Assays for analyzing gene-specific CpG sites
PyroMark CpG Assays enable methylation analysis of specific targets across the genome. Our database now contains over 84,000 predesigned assays covering more than 80% of gene-specific CpG islands for the human, mouse, and rat genomes. These assays are designed using a carefully tailored algorithm that generates optimal assay design based on bisulfite converted DNA. As such, these fast and easy-to-order assays minimize time spent on assay optimization and maximize analysis success. PyroMark CpG Assays expand the streamlined Pyrosequencing workflow to enable methylation analysis of virtually any CpG island in the human, mouse, or rat genome.
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Assays for methylation array validation
Hybridization arrays enable high-throughput discovery of variations in DNA methylation, but the results must be experimentally verified to confirm that the correct variations are identified. Pyrosequencing is a cost-effective technique highly suited for this purpose, providing quantitative analysis of short- to medium-length sequences with high accuracy. A recent article from Roessler and colleagues describes analysis of genomewide aberrations in DNA methylation using the Infinium HumanMethylation450K BeadChip array, followed by quantification of methylation levels for 692 individual CpG sites from 62 different genes using Pyrosequencing (1). The results of this cross-validation study showed very good concordance between both methods, confirming Pyrosequencing as a method highly suited for validation of array results.
QIAGEN now offers predesigned PyroMark CpG Assays for validating results from HumanMethylation450K BeadChip experiments. These new predesigned assays for methylation array validation offer a convenient way to verify experimental results, and determine exact methylation levels of CpG sites of interest, without the need to design a separate assay for each individual target of interest. Our dedicated search page lets users simply enter the CpG loci identification numbers (CG#) of interest to easily search our assay database and order the ready-to-use PyroMark CpG Assays. If an assay is not already available for a particular target, one will be automatically designed within just a few minutes using our dedicated algorithm and made available in our database.
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1. Roessler, J., Ammerpohl, O., Gutwein, J., Hasemeier, B., Anwar, S.L., Kreipe, H., and Lehmann, U. (2012) Quantitative cross-validation and content analysis of the 450k DNA methylation array from Illumina, Inc. BMC Research Notes. 5:210.