We have developed a method that enriches for methylated cytosines by capturing 
the fraction of bisulfite-treated DNA with unconverted cytosines. The method, 
called streptavidin bisulfite ligand methylation enrichment (SuBLiME), involves 
the specific labeling (using a biotin-labeled nucleotide ligand) of methylated 
cytosines in bisulfite-converted DNA. This step is then followed by affinity 
capture, using streptavidin-coupled magnetic beads. SuBLiME is highly adaptable 
and can be combined with deep sequencing library generation and/or genomic 
complexity-reduction. In this pilot study, we enriched methylated DNA from 
Csp6I-cut complexity-reduced genomes of colorectal cancer cell lines (HCT-116, 
HT-29 and SW-480) and normal blood leukocytes with the aim of discovering 
colorectal cancer biomarkers. Enriched libraries were sequenced with SOLiD-3 
technology. In pairwise comparisons, we scored a total of 1,769 gene loci and 33 
miRNA loci as differentially methylated between the cell lines and leukocytes. 
Of these, 516 loci were differently methylated in at least two promoter-proximal 
CpG sites over two discrete Csp6I fragments. Identified methylated gene loci 
were associated with anatomical development, differentiation and cell signaling. 
The data correlated with good agreement to a number of published colorectal 
cancer DNA methylation biomarkers and genomic data sets. SuBLiME is effective in 
the enrichment of methylated nucleic acid and in the detection of known and 
novel biomarkers.