Bisulfite pyrosequencing is a sequencing-by-synthesis method used to quantitatively determine the methylation of individual CG cytosines from PCR amplicons of a region up to 115 bases in length. The procedure relies on prior bisulfite conversion of all potentially methylated CG cytosines to either cytosine (methylated) or thymine (unmethylated) and involves the stepwise incorporation of deoxynucleotide triphosphates into the growing strand of nascent DNA. The incorporation of these dNTPs results in the proportional release of pyrophosphate, which is converted into ATP to aid in a subsequent conversion of luciferin to oxyluciferin. The amount of light released in the process is proportional to the number of nucleotides incorporated, and the procedure provides a quantitative portrait of the methylation profile for the amplicon in question.
Key wordsPCR Pyrosequencing Sequencing-by-synthesis Methylation Epigenetics
We gratefully acknowledge Zack Davenport for his contributions to the artwork. We thank Allison Barratt and Carole Grenier for critical reading of the manuscript. This work was supported by Department of Defense grant W81XWH-11-1-0469, NIH grants R01DK085173 and R01CA142983, and the Gail Parkins Ovarian Cancer Awareness Fund.
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