A Primer Design Algorithm for Global Analysis of CpG Methylation

Global DNA methylation changes have been documented to occur during normal development, aging and disease progression. Genome-wide levels of methylation are commonly inferred from the average level of methylation of large sets of repetitive elements. Alu repeats, in particular, have been exploited in a variety of approaches to monitor genome-wide losses in CpG methylation. Alu elements encompass over 25% of the CpG dinucleotides in the human genome. However, due to the high frequency of deamination of methylated cytosines in repetitive elements occurring in the germline, the sensitivity to detect methylation changes based upon their methylation levels is greatly compromised. In addition, the number of repeats that were targeted in previous investigations for ascertainment of genome-wide levels of methylation could not be accurately estimated. In this study, we developed and implemented an algorithm to design highly specific primers altogether targeting 617,817 CpG sites in over 45,000 Alu elements. In combination with the pyrosequencing technology, these primers were shown to yield highly sensitive and reproducible results.