Early Detection of Cancer
From a clinical perspective, DNA methylation changes in cancer represent an attractive therapeutic target as epigenetic alterations are, in principle, more readily reversible than genetic events. Another area of great promise is in molecular diagnostics and early detection. Cancer-specific DNA methylation patterns can be detected in tumor-derived free DNA in the bloodstream and in epithelial tumor cells shed into the lumen.
The complexity of varying distributions of methylated cytosines across the approximately 50 million CpG dinucleotides of each diploid mammalian genome in a DNA sample derived from a heterogeneous tissue sample is a diagnostic dream and an analytical nightmare. Methylation profiles yield information on the methylation status across many sites in the genome, providing a unique approach to genome-wide molecular diagnostics.
Most microarray-based DNA methylation analysis platforms rely on methylation-sensitive restriction enzyme digestion or on enrichment using methylcytosine antibodies or methyl-binding domain protein columns. These methods can assay a relatively large number of features (loci) when combined with high-density tiling or promoter microarrays, but are limited in their sample throughput capacity, and generally require large amounts of high-quality input DNA. Most other DNA methylation analysis techniques use bisulfite-based conversion of unmethylated cytosines into uracils to turn epigenetic differences into sequence-based information. Many bisulfite-based techniques such as methylation-specific PCR (MSP) and MethyLight can accommodate large numbers of samples, but are limited in the number of loci that can be interrogated.
By relying on the collective address-tagged amplification of 1,536 CpG sites and hybridization on a BeadArray™ substrate, the GoldenGate DNA methylation analysis platform recently introduced by Illumina (www.illimina.com) retains the high sample throughput and large number of loci that can be interrogated simultaneously (Table).
The methylation platform assesses the methylation status of up to 1,536 independent CpG sites per assay across 96 samples. It advances methylation research and biomarker discovery with high reproducibility (average r2 Ž 0.98), flexible content design, strong correlation to MSP and high sensitivity to detect small differences in methylation status between biological samples. Differential methylation analysis can be accomplished in less than one week from bisulfite conversion to data analysis.
To assay DNA methylation status, cytosine is converted to uracil with bisulfite treatment. The fact that 5-methylcytosine is unreactive under the same conditions is important for the assay’s utility. The standard assay used for methylation profiling is an adaptation of the GoldenGate Assay for genotyping (Figure 1). Bisulfite-converted DNAs are biotinylated and immobilized on paramagnetic beads. Query oligonucleotides are hybridized to the DNA and then washed to remove excess or mishybridized oligonucleotides. The hybridized oligos are then extended and ligated to create amplifiable templates. The PCR that follows uses fluorescently labeled universal PCR primers. Methylation status of the interrogated CpG sites is determined by comparing the ratio of the fluorescent signal from the methylated allele to the sum of the fluorescent signals of both methylated and unmethylated alleles.