FIG. 2.

MAR assays. We extracted 10⁵ nuclei from a tissue sample with 2 M NaCl. The resulting nuclear halos were digested with restriction enzymes (A). Matrix-bound DNA (pellet, P) was isolated from loop DNA (supernatant, S) by ultrafiltration (see Materials and Methods). The relative enrichment of target sequences in the pellet fraction was determined by real-time quantitative PCR. For allele-specific MAR assays, primers were designed on either side of polymorphic restriction sites and quantifications were performed on DNA from the pellet fraction undigested or digested with the polymorphic enzyme (see Materials and Methods). Alternatively, for precise mapping of nuclear matrix attachments, nuclear halos were digested with DNase I before performing real-time PCR quantifications on the pellet fraction (B). Pictures show nuclei, nuclear halos, and restriction enzyme-treated halos stained with Hoechst. Scale bars, 10 μm.