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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. Introduction Methylation at the 5′ position of cytosines yielding 5-methyl-cytosine (5-mC) is an epigenetic mark that regulates the expression of genes.

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DNA methylation is catalyzed by DNA methyltransferases, is a dynamic process and, along with other epigenetic regulators, guides differentiation and development. While the large majority of cytosine methylation in vertebrate genomes is found in the context of symmetric CpG dinucleotides, methylation at CpNpG sites is also common in plant genomes and recent data has indicated the presence of significant non-CpG methylation in mammalian embryonic stem cells.

Presently, there is great interest in establishing correlations between phenotype and DNA methylation state and in discovering epigenetic biomarkers of disease. Most effort in studying and comparing methylomes has been directed toward identifying differentially methylated regions (DMRs) between human neoplastic and normal cells. It is well established that aberrant epigenetic regulation observed in cancer is manifested as global changes that alter chromatin packaging and localized changes at gene promoters, which influence the transcription of genes.

Relative to normal cells, cancerous cells exhibit genome-wide hypomethylation in large blocks, juxtaposed with hypermethylation at select gene promoters. - Aberrant methylation can drive carcinogenesis through the silencing of tumor suppressor genes and erosion of chromosome stability., Accordingly, there is much interest in detecting DMRs between normal and cancerous cells, as these might be driving cancer progression. The current DNA methylation gold standard technique for quantifying methylation at nucleotide-base resolution across a genome is whole-genome bisulfite shotgun sequencing (WGBS).

Limba Romana Manual Pentru Studentii Straini Grigore Brancus Pdf. Unfortunately, WGBS is still prohibitively expensive for most laboratories, particularly when many replicates are required; significantly, a high fraction of reads contain no methylated cytosines. To reduce cost and/or enable analysis of higher sample numbers, typically some form of genomic complexity-reduction and/or DNA methylation enrichment procedure followed by microarray or deep sequencing is used. Broadly, there are three main methods used to derive methylome data: bisulfite sequencing, affinity purification of methylated DNA and enzymatic restriction using enzymes with methylation sensitivity. While bisulfite conversion of unmethylated cytosines offers nucleotide level resolution of the methylome, it results in a loss of DNA “information content,” makes deep sequencing technologies more error-prone and effectively doubles the genome size—as Watson and Crick strands no longer are complementary. This poses challenges in the alignment of short bisulfite DNA reads back to large genomes, both from the loss of unique alignments and the large increase in alignment candidates per read. The computational burden can be reduced significantly with genomic complexity-reduction, usually implemented via restriction enzyme-based methods. In these instances, the methylome is sampled around restriction sites.