Figure 2.

A schematic outline to identify differentially methylated genes using promoter microarrays. Genomic DNAs from secondary palates (gestation days 12–14) were isolated and fragmented by sonication. Sonicated DNA was incubated with MBD2b, a methyl-CpG binding protein (Active Motif, Inc., Carlsbad, CA), for enrichment of methylated genomic fragments. The enriched DNA fragments from all three gestational days were amplified by whole genome amplification. Genomic DNAs not subjected to methylation enrichment were used as controls. Control DNA, which was labeled with Cy3, and experimental DNA, which was labeled with Cy5, were hybridized to NimbleGen 2.1 M mouse promoter microarrays (Roche NimbleGen, Madison, WI). Nine microarrays representing three biologic replicates on each of gestation days 12, 13, and 14 were probed. After hybridization and washing, the microarray chips were scanned and the accrued data were analyzed using biostatistical methods (Seelan et al., unpublished data).