Pyrosequencing Methylation Analysis

Abstract

Pyrosequencing, a real-time sequencing technology, is considered a “gold standard” for quantitative allele quantification at single base resolution. Quantitative bisulfite Pyrosequencing determines DNA methylation level by analyzing artificial “C/T” SNPs at CpG sites within a specific Pyrosequencing assay. The bisulfite Pyrosequencing methylation assay design is DNA strand specific and the primer design should not contain any CpG sites and should be free of high-frequency mutations. Additionally Pyrosequencing assays must be tested for preferential amplification during bisulfite PCR to ensure the sequencing quantification accuracy and reproducibility. Pyrosequencing analysis gives a reproducible measurement of average methylation at several CpG sites within the Pyrosequencing assay directly from a PCR product, rapidly and accurately for many samples at a time. It is therefore well suited for clinical research, validation of whole-genome methylation screening results, and global methylation analysis using repetitive elements including LINE-1, Alu, and Sat2. Pyrosequencing reproducibility and accuracy result in low measurement variance, thereby increasing the likelihood of early detection of small changes in methylation levels that may become apparent in response to treatment. For example, the high reproducibility of the LINE-1 assay is important for detecting the relatively small daily changes in methylation levels associated with hypomethylation. This enables detection of differences in patterns between normal and disease tissue such as in tumor suppresser genes, and to determine global methylation changes in response drug treatments. Relatively low cost and easy automation allows the researcher to increase the experiment’s sample population to detect trends that would otherwise not have a sufficient sampling basis for statistical significance.