PyroMark CpG Assays

Primer design optimized for high performance

Highly specific and unbiased amplification is especially important for methylation analysis by Pyrosequencing. Because bisulfite converted DNA is a less complex template, it can be challenging to amplify specifically and at high yields, but with predesigned PyroMark CpG Assays, this is not a problem. The PCR and sequencing primers included 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). The high-performing assays eliminate the need for tedious assay optimization and shorten the time to reliable results.

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 (see figure Analysis of ESR1).

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. We now offer predesigned PyroMark CpG Assays for validating results from HumanMethylation450K and MethylationEPIC 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 manually 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 ready-to-use PyroMark CpG Assays.

The advantages of GeneGlobe

QIAGEN's GeneGlobe Web portal is a one-stop online interface that grants access to our extensive range of assay technologies in a gene- or pathway-specific context. GeneGlobe brings together state-of-the-art search and design algorithms, up-to-date sequence data, and our innovative technologies to facilitate discovery. Simply enter a search term for the gene or pathway of interest, and let GeneGlobe show you how to advance your research in epigenetics, or any other area of interest (see figure Ready-to-use methylation assays).

PyroMark CpG Assays include PCR and sequencing primers for quantification of DNA methylation by Pyrosequencing. A biotinylated PCR product is generated from the bisulfite converted DNA template and is bound to streptavidin-coated Sepharose beads. The beads are captured with the vacuum tool on the PyroMark Vacuum Workstation, where they are thoroughly washed and subsequently denatured, generating single-stranded DNA suitable for Pyrosequencing. This template DNA is released into the Pyrosequencing reaction plate containing the sequencing primer provided with PyroMark CpG Assays. After primer annealing, the plate is placed into the PyroMark instrument, and the sequencing reaction is started.

PyroMark CpG Assays enable highly accurate, quantitative methylation analysis of gene-specific human, mouse, and rat CpG sites as well as targets from the HumanMethylation450K and the MethylationEPIC BeadChip for epigenetics research.