Figure 1

Overview and pre-microarray performance of MBD-chip. A, Overview of MBD-chip. Genomic DNA is: i) fragmented (in this case with restriction enzymes), ii) enriched for methylated DNA using MBD2-MBD-magnetic beads, and iii) amplified, fragmented, labeled and hybridized to tiling microarrays. Comparison with a total input fraction allows identification of methylated regions. B, Degree of MBD2-MBD enrichment is non-linearly proportional to the number of methylated CpGs. WBC DNA was methylated at 0, 6, 10 or 37 CpG sites within an R.AluI restriction fragment within the GSTP1 promoter by treatment with M.HpaII +/- M.HhaI or with M.SssI or no enzymes. The degree of MBD2-MBD enrichment compared to mock (no MBD control), as measured by qPCR, was related to the number of methylated CpGs. ND, not detectable. C, The MBD-chip process enriches DNA with high density of methylated-CpGs. DNA from LNCaP and PrEC cells was completely methylated with M.SssI or left untreated. MBD2-MBD enrichment at regions in HBB and GSTP1 promoters, as examined by qPCR, are shown. The CpG density (Low, indicates < 5 CpGs per kbp and high indicates > 20 CpGs per kbp) and known degree of methylation (as determined by the Infinium 27K DNA methylation platform for HBB (unpublished data; Yegnasubramanian S and Haffner MC, 2011) and by bisulfite sequencing for GSTP1 [9]) are indicated. Schematics of each region are annotated with position of CpGs (vertical hashes), transcriptional start sites (yellow arrow), and amplification primers (red arrows). The start and end positions are with respect to transcriptional start sites.