Methylation profiling using degenerated oligonucleotide primer-PCR specific for genome-wide amplification of bisulfite-modified DNA.

Abstract DNA methylation is one of the essential epigenetic processes that play a role in regulating gene expression. Aberrant methylation of CpG-rich promoter regions has been associated with many forms of human cancers. The current method for determining the methylation status relies mainly on bisulfite treatment of genomic DNA, followed by methylation-specific PCR (MSP). The difficulty in acquiring a methylation profiling often is limited by the amount of genomic DNA that can be recovered from a given sample, whereas complex procedures of bisulfite treatment further compromise the effective template for PCR analysis. To circumvent these obstacles, we developed degenerated oligonucleotide primer (DOP)–PCR to enable amplification of bisulfite-modified genomic DNA at a genome-wide scale. A DOP pair was specially designed as follows: first 3′ DOP, CTCGAGCTGHHHHHAACTAC, where H is a mixture of base consisting of 50% A, 25% T, and 25% C; and second 5′ DOP, CTCGAGCTGDDDDDGTTTAG, where D is a mixture of base consisting of 50% T, 25% G, and 25% A. Our results showed that bisulfite-modified DNAs from a cell line, cord blood cells, or cells obtained by laser capture microdissection were amplified by up to 1000-fold using this method. Subsequent MSP analysis using these amplified DNAs on nine randomly selected cancer-related genes revealed that the methylation status of these genes remained identical to that derived from the original unamplified template.