Methylation Status of Individual CpGs
Bisulfite Sequencing

Assay includes: Primer design, isolation of DNA from cells, bisulfite conversion of DNA, PCR amplification and gel analysis of PCR products, gel purification of PCR products, cloning of PCR products, amplification of cloned inserts (8 clones per sample per region), sequencing, data analysis

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DNA methylation—a chemical modification of DNA that acts as an on-off switch for genes—occurs through the reversible addition of methyl groups at cytosines within CpG dinucleotides. DNA methylation can prevent binding of transcription factors to their binding motifs and can also recruit proteins that promote transcriptional repression. Identifying methylation patterns at specific genes of interest will provide a mechanistic understanding of how genes are regulated. Additionally, identification of differential methylation patterns are useful as diagnostic and prognostic biomarkers.

Bisulfite Sequencing is the highest-resolution assay available for determining the methylation status of individual CpG dinucleotides. Genes known to be regulated in disease or upon drug treatment can be interrogated to identify differences in cytosine methylation patterns that correlate with the disease state or treatment. The method involves bisulfite treatment of DNA, which converts unmethylated cytosines to uracil while methylated cytosines are resistant to conversion. Upon sequencing, unmethylated cytosines are presented as Ts, while methylated cytosines remain as Cs. Genpathway’s Bisulfite Sequencing Service is also a useful companion assay to MethylPath™, enabling high-resolution validation of methylation differences identified on a genome-wide platform.

Example of Bisulfite Sequencing Data and Analysis >

Genpathway Sample Preparation for DNA Methylation Assays >