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J Biomol Tech. 2007 February; 18(1): 17.
PMCID: PMC2292055

P47-M Comparison of Pyrosequencing and Fluorescent Dideoxynucleotide Sequencing for Measurement of CpG Methylation


In mammals, cytosine methylation at CpG sites is an important epigenetic mechanism for altering gene expression in normal and cancer cells. Sequencing of PCR products from bisulfite-treated genomic DNA provides the most direct measurement of methylation at individual cytosines. To compare the relative ability of pyrosequencing and fluorescent dideoxynucleotide sequencing to accurately quantify cytosine methylation, I designed primers for amplification of a 197-bp region within the IGF2 gene. Two sets of primers were synthesized: (1) a set containing a 5′-biotin on the reverse primer (used for streptavidin purification of the template strand prior to pyrosequencing) and (2) a set containing the M13 forward and M13 reverse primer sequences as 5′ extensions of the forward and reverse PCR primers, respectively. Only the first set was used to generate PCR products for pyrosequencing; both sets were used to make amplicons for fluorescent DNA sequencing. Following PCR amplification of eight bisulfite-treated human genomic DNAs, templates were purified and subjected to both DNA sequencing technologies. Pyrosequencing was able to measure percent methylation at 7 CpG sites, while the fluorescent chemistry was able to interrogate up to 12 sites, due in part to its longer read length. The percent methylation measured by each technology differed for a given CpG site and genomic DNA: the value obtained by pyrosequencing was usually 10–15% less than that obtained by the fluorescent chemistry. In order to determine which chemistry measures percent methylation more accurately, experiments with known ratios of fully methylated and unmethylated templates are underway.

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