Pyrosequencing is a DNA sequencing-by-synthesis technique that can quantitatively detect single-nucleotide polymorphisms (SNPs). With pyrosequencing, the level of DNA methylation can be calculated according to the ratio of artificial cytosine/thymine SNPs produced by bisulfite conversion at each CpG site. This analysis method provides a reproducible and accurate measurement of methylation levels at CpG sites near sequencing primers with high quantitative resolution. DNA methylation plays an important role in mammalian development and cellular physiology; alterations in DNA methylation patterns have been implicated in several common diseases as well as cancers and imprinting disorders. Evaluating DNA methylation levels via pyrosequencing is useful for identifying biomarkers that could help with the diagnosis, prognosis, treatment selection, and onset risk assessment for several diseases. We describe the principles of pyrosequencing and detail a bisulfite pyrosequencing protocol based on our experience and the PyroMark Q24 User Manual.
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